RESEARCH ARTICLE – Pharmaceutical Biotechnology

Protein Aggregation and Particle Formation in Prefilled Glass Syringes ALANA GERHARDT,1 NICOLE R. MCGRAW,1 DANIEL K. SCHWARTZ,1 JARED S. BEE,2 JOHN F. CARPENTER,3 THEODORE W. RANDOLPH1 1

Department of Chemical and Biological Engineering, University of Colorado – Boulder, Boulder, Colorado Formulation Sciences, MedImmune, Gaithersburg, Maryland 3 Department of Pharmaceutical Sciences, University of Colorado – Denver, Aurora, Colorado 2

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 protein molecules 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 to silicone oil–water interfaces (siliconized syringe walls), air–water interfaces (air bubbles), and agitation stress (occurring during end-overend rotation). Using flow microscopy, particles (≥2 ␮m diameter) were detected under all conditions. The highest particle concentrations were found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oil droplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism of particle generation in PFS in which capillary forces at the three-phase (silicone oil–water–air) contact line remove silicone oil and gelled protein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil–water C 2014 Wiley Periodicals, Inc. and the interfaces, air–water interfaces, and agitation in the generation of particles in protein formulations.  American Pharmacists Association J Pharm Sci 103:1601–1612, 2014 Keywords: PFS; silicone oil; microparticles; protein formulation; protein aggregation; image analysis; adsorption; monoclonal antibody

INTRODUCTION Glass prefilled syringes (PFS) are often the packaging method of choice for therapeutic protein products. Their many benefits include reduced contamination risk, minimal overfill, dose accuracy, ease of administration, and improved patient compliance.1 Thus, it is not surprising that PFS are one of the fastest growing markets in the drug delivery sector, and their use is expected to increase further with the growing demand for injectable biologic drugs.1 Prefilled syringes serve as both the delivery device and the storage container for protein therapeutics. During a product’s shelf life (typically 2 years), therapeutic protein molecules are exposed to a variety of interfaces in PFS, including silicone oil–water interfaces and air–water interfaces. The silicone oil– water interface is present because silicone oil is commonly used as a lubricant to reduce and smooth the force required for injection. In typical PFS, levels of siliconization are approximately 0.4–1.0 mg silicone oil per 1 mL syringe.2 The air–water interface is introduced in the syringe because of an air bubble that remains as a consequence of the syringe filling and stoppering process. Several recent studies have focused on how proteins may be affected by exposure to these interfaces. In particular, protein adsorption to both air–water and silicone oil–water inCorrespondence 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 upon request 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 and particle formation.3–9 The presence of particles of any kind in a protein formulation is a major concern. Both proteinaceous particles and particles shed during manufacturing and storage (i.e. glass, stainless steel, and silicone oil) may be present in therapeutic protein products.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 the number of particles between 0.1 and 10 :m in protein formulations, as particles in this size range may be the most immunogenic and are considered a critical quality attribute.12 With new technologies, the ability to quantify and characterize particles in the 0.1–10 :m size range has significantly improved,10,14–16 and as a result, several recent studies have focused on identifying the extent to which these particles arise in protein therapeutic products and their impact on product quality, safety, and immunogenicity.5,17–21 For example, Barnard et al.20 suggest a link between the presence of aggregates and particles in interferon-$ products and the production of neutralizing antibodies in patients. In contrast, Lubiniecki et al.21 found that although switching a protein drug product from vials to PFS resulted in a slight and statistically significant increase in the levels of subvisible particles, there were no detectable differences in patient responses in clinical trials between products in vials versus products in PFS. In addition to the exposure to interfacial stresses, protein formulations are also subject to various transportation-associated stresses such as agitation. Studies of protein formulations that were agitated in the presence of a silicone oil emulsion have

Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

1601

1602

RESEARCH ARTICLE – Pharmaceutical Biotechnology

shown large losses of monomeric protein due to the aggregation and not simply adsorption to the interface.3,22 Other recent studies have detected significant particle formation in protein formulations agitated in siliconized syringes.5,9 However, some researchers have suggested that the particles formed in stressed PFS samples are composed of only silicone oil droplets and that there is minimal impact on protein aggregation from the presence of silicone oil, in particular if the protein formulation contains surfactant.5,21,23 However, even particles composed 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 act synergistically 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 hydrophobic interfaces, such as the silicone oil–water interface and the air–water interface.3,7,25,26 Upon adsorption to hydrophobic interfaces, many proteins form viscoelastic gel layers.27–29 Disruption of the protein gel layer results in the release of protein aggregates and particles into the bulk solution.6,7 Further work is needed to evaluate the role of the silicone oil–water interface in generating protein aggregates and particulates and to determine how interfaces and agitation combine synergistically to compromise the quality of therapeutic protein formulations. Therefore, the objective of this study was to determine the mechanism by which silicone oil–water interfaces, air–water interfaces, and agitation work together to induce particle generation in protein formulations. The effects of these three factors were evaluated in two different formulations (containing either lysozyme or a monoclonal antibody) in PFS. Protein formulations were incubated quiescently or with end-over-end rotation in siliconized or unsiliconized glass syringes, both in the presence and absence of an air bubble. Under certain conditions, glass beads were added to the syringes to add bulk shear forces without the addition of an air bubble. The number of particles generated in each incubation condition was counted using flow microscopy, which also recorded images of the observed particles. Because we wanted to observe the effect of the air–water interface and the silicone oil–water interface on the protein formulation, the formulations were prepared without surfactant present. In addition, we measured the interfacial tensions of the silicone oil–water interface and the air–water interface with protein adsorbed and the contact angle of a protein solution on a siliconized surface in order to estimate the forces acting inside a PFS.

MATERIALS AND METHODS Materials Humanized IgG1 monoclonal antibody (molecular weight 146 kDa), here denoted as “3M”, was provided by MedImmune (Gaithersburg, Maryland).30 The antibody was obtained at a stock concentration of 150 mg/mL in 10 mM L-histidine at pH 6. For consistency with previous work,3 3M formulations were prepared in 10 mM L-histidine pH 5. Lysozyme from chicken egg white (molecular weight 14.3 kDa) with ≥90% purity was purchased as a lyophilized powder from Sigma–Aldrich (St. Louis, Missouri). Silicone oil (Dow Corning 360, 1000 cSt) was of medical grade and purchased from Nexeo Solutions (Denver, Colorado). All buffer salts were of reagent grade or higher. Bradford reagent (Brilliant Blue G in phosphoric acid and methanol) Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

was obtained from Sigma–Aldrich. All solutions were prepared in deionized water filtered with a 0.22 :m Millipore filter (Billerica, Massachusetts). The syringes used in the incubation studies were BD Hypak SCF 1 mL long 27G1/2 (BD MedicalPharmaceutical Systems, Franklin Lakes, New Jersey). 3M Adsorption to Surfaces To compare the surface coverage of 3M on glass with the previously measured surface coverage of 3M on silicone oil,3 glass particles were prepared from Type I glass vials (5cc, Type 1 Glass, USP/PhEur, nontreated; Alcan Packaging, Syracuse, Nebraska) as described in Bee et al.31 The particles were sieved through a 45 :m screen before use. A suspension of 100 mg/mL glass microparticles in 10 mM L-histidine buffer pH 5 was prepared for use in the adsorption studies. The particle surface area and the particle size distribution of the glass suspension were measured as previously described.31 A bulk depletion method similar to that described by Gerhardt et al.3 was used to measure the amount of 3M adsorbed to glass surfaces. Aliquots of 3M stock solution (3M concentration 0.73 mg/mL) ranging in volume from 0.00 to 0.10 mL were mixed with a suspension of glass microparticles in 10 mM L-histidine to achieve a total volume of 0.5 mL. The samples were incubated end-over-end at 8 rpm for 1 hour at room temperature to allow the protein to adsorb to the suspended glass microparticles. After incubation, the vials were centrifuged at 12,000g for 30 min to separate the glass microparticles from the aqueous phase, and approximately 300 :L of the supernatant was removed and transferred to 0.6 mL microcentrifuge vials. Following previously described methods, a modified Bradford assay was performed to determine the bulk 3M concentration of each sample and subsequently the amount of 3M adsorbed, and the data were fit to a Langmuir isotherm to provide an estimate of the monolayer surface coverage of 3M on glass.3 Removal of Silicone Oil from Syringes As controls, some incubation studies were conducted in unsiliconized syringes. To remove the silicone oil coating, syringes were cleaned as follows. A 1% solution of Micro-90 (International Products Corporation, Burlington, New Jersey) was pipetted in and out of the syringes four times. This was followed by a rinse with deionized water. Then, hexane (ACS grade; EMD, Billerica, Massachusetts) was pipetted in and out of the syringes five times, and the syringes were allowed to air dry at room temperature. Finally, the syringes were submerged in piranha solution (70% sulfuric acid:30% hydrogen peroxide) for 1 hour (with the needle facing up and out of the solution) and then rinsed with deionized water and dried with nitrogen gas. Caution: Piranha solution is extremely corrosive and should be handled with extreme care. Contact angle measurements on treated glass slides were used to verify that this cleaning procedure successfully removes silicone oil from glass. First, the contact angle of a 1 :L droplet of 0.2 :m-filtered deionized water on a bare glass slide was measured using an Artcam-130MI-BW camera (Artray Company, Ltd., Tokyo, Japan). The contact angle of water on the slide was calculated based on the shape of the droplet using the FTA32 Video 2.0 software (First Ten Angstroms, Portsmouth, Virginia). Then, slides were incubated overnight in a 3% solution of DC 360 medical fluid (1000 cSt; Dow Corning, Midland, Michigan) in toluene to coat them with silicone oil. After excess DOI 10.1002/jps.23973

RESEARCH ARTICLE – Pharmaceutical Biotechnology

liquid was removed from the surface of the glass slide, the remaining toluene was allowed to evaporate, leaving a thin, uniform coating of silicone oil on the glass slide. Subsequently, the above-described cleaning procedure was performed to remove the silicone oil coating, and the contact angle of a 1 :L droplet of deionized water was measured on the cleaned surface. The contact angles measured before and after coating and cleaning were identical within experimental error (data not shown). In addition, the difference between the hydrophobic siliconized syringe surface and the hydrophilic cleaned bare glass syringe surface was confirmed by filling each syringe with 1 mL of deionized water and visually comparing the shape of the menisci (Supplemental Information Fig. S1).

1603

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

Incubation of Protein Formulations in PFS Two formulations of proteins were prepared for incubation in syringes: 1 mg/mL 3M in 10 mM L-histidine pH 5 and 1 mg/mL lysozyme in 10 mM potassium phosphate pH 7.2. For incubations with an air bubble, 1.26 mL of the formulation was pipetted into the syringe, and the syringe was stoppered, which created a headspace containing 30 :L of air. For incubation conditions with no air bubble, the syringe was stoppered such that no air bubbles remained in the syringe. For incubation conditions with beads, two 4 mm glass beads were added to the syringe, which was then filled with protein formulation and stoppered such that no air bubbles remained in the syringe. Triplicate syringes were prepared for each incubation condition at each time point. For incubation conditions with agitation, the syringes were rotated end-over-end at 1.5 rpm at room temperature. For stationary incubation conditions, the syringes were incubated horizontally on the bench top at room temperature. Counting of Particles in Incubated Protein Formulations At each time point, the syringes were unstoppered, and the formulations were removed from the flanged end using a transfer pipet. The protein formulation was not ejected using the syringe needle to avoid the generation of particles due to the plunger movement along the syringe barrel. For each sample, particles between 2 :m and 2 mm (equivalent spherical diameter) were counted using a Fluid Imaging Technologies Benchtop FlowCAM (Scarborough, Maine). The FlowCAM was fitted with a FC100 flow cell, a 10× objective and collimator, and a 0.5 mL syringe. The gain and flash duration were set such that the average intensity mean of the image was consistently between 180 and 200. A sample volume of 0.2 mL was analyzed for each sample at a flow rate of 0.145 mL/min. Particle counts were normalized by dividing the number of particles per sample by the total volume imaged per sample to obtain the particle concentration (#/mL). In addition to the samples incubated in syringes, buffer solutions and protein formulations not incubated in syringes were also analyzed by FlowCAM . R

R

Contact Angle and Interfacial Tension Measurements The contact angle (measured through the liquid) of a 1 :L droplet of 1 mg/mL 3M formulation was measured on a siliconized glass slide using the method described above. Measurements were made on two different slides with three droplets measured on each slide. All measurements were averaged, and the standard deviation was reported. Interfacial tension measurements were conducted using the pendant drop technique.32 A camera (described above) was used DOI 10.1002/jps.23973

RESULTS 3M Adsorption to Glass The surface coverage of 3M on glass was plotted as a function of the bulk 3M concentration and fit to a Langmuir isotherm model (data not shown). The amount of adsorbed 3M to form monolayer 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 formulations and in a buffer solution incubated in PFS under all conditions (Fig. 1). A buffer solution incubated in agitated, siliconized syringes, with or without an air bubble, contained low levels of particles that did not change appreciably over the course of 2 weeks (Fig. 1a). The observed particles were spherically shaped and, thus, assumed to be silicone oil droplets (Fig. 2a). In addition, a similarly low level of particles was observed in 3M formulations incubated in siliconized syringes that were not agitated (Fig. 1b), and the particle concentrations did not change over time. Both silicone oil droplets and protein aggregates were observed in this condition (Fig. 2b). The greatest number of particles was detected in 3M formulations incubated in agitated, siliconized syringes with an air bubble (Fig. 1d; closed symbols). In these formulations, the particle concentrations increased steadily over time. Images collected during flow microscopy suggested that these particles typically 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 resulting particle concentrations were two orders of magnitude lower than those observed in siliconized syringes with an air bubble (Fig. 1d; open symbols), and the particle concentrations were invariant over time. In the absence of an air bubble, the number of particles detected was not appreciably higher than the number of particles detected in the incubated buffer solutions and in the absence of agitation. In some siliconized syringes without air bubbles, glass beads were added to introduce shearing in the syringes without the addition of an air–water interface. In otherwise identical conditions (i.e. 3M formulations in agitated, siliconized syringes), particle counts were lower in the presence of glass beads than in the presence of an air bubble (Fig. 1d; cross symbols). In the presence of the glass beads, the particle concentrations also increased with time, and, the images of particles showed both silicone oil droplets and aggregated protein (Fig. 2e). Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

1604

RESEARCH ARTICLE – Pharmaceutical Biotechnology

Figure 1. Particle concentrations in 3M formulations and buffer solutions incubated in PFS as a function of time. Open symbols correspond to syringes incubated with no air bubble, closed symbols correspond to syringes incubated with an air bubble, and cross symbols correspond to syringes 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 formulations incubated in agitated, unsiliconized syringes with no air bubble were comparable to those observed in 3M formulations incubated in agitated, siliconized syringes with no air bubble (Figs. 1c and 1d; open symbols). The addition of an air bubble to 3M formulations incubated in agitated, unsiliconized syringes slightly increased the number of particles detected (Fig. 1c; closed symbols). However, the particle concentrations in 3M formulations incubated in agitated, unsiliconized syringes with an air bubble remained an order of magnitude lower than the particle concentrations in 3M formulations incubated in agitated,

Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

siliconized syringes with an air bubble (Figs. 1c and 1d; closed symbols). The images of particles in 3M formulations incubated in unsiliconized syringes showed only protein aggregates (Fig. 2c). In summary, a baseline level of particles was observed under all incubation conditions. The particle concentrations increased above this baseline level in protein formulations incubated in agitated, unsiliconized syringes with an air bubble and in agitated, siliconized syringes with glass beads, but the largest particle concentrations were observed in protein formulations incubated in agitated, siliconized syringes with an air bubble.

DOI 10.1002/jps.23973

RESEARCH ARTICLE – Pharmaceutical Biotechnology

1605

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

DOI 10.1002/jps.23973

Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

1606

RESEARCH ARTICLE – Pharmaceutical Biotechnology

Figure 3. Particle concentrations in lysozyme formulations and buffer solutions incubated in PFS as a function of time. Open symbols correspond to syringes incubated with no air bubble, closed symbols correspond to syringes incubated with an air bubble, and cross symbols correspond to syringes incubated with no air bubble but with glass beads. The particle concentrations in a buffer solution (solid black line) and in a lysozyme solution (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 PFS Particles 2 :m or greater were detected in lysozyme formulations and in a buffer solution incubated in PFS under all conditions (Fig. 3). The trends in particle concentration were similar in lysozyme formulations as in 3M formulations, and the images of particles in lysozyme formulations were similar to those for 3M formulations (Supplemental Information Fig. S3). The lowest levels of particles were detected in a buffer solution in agitated, siliconized syringes (Fig. 3a), with slightly higher levels in lysozyme formulations incubated in siliconized syringes that were not agitated (Fig. 3b). Lysozyme formulations incubated in agitated, unsiliconized syringes also contained low Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

levels of particles (Fig. 3c), but the particle concentrations did not change over time. Lysozyme formulations incubated in agitated, siliconized syringes with an air bubble contained the greatest number of particles (Fig. 3d), and the particle concentrations increased with time. As with 3M formulations, lysozyme formulations incubated in agitated, siliconized syringes with no air bubble exhibited particle concentrations 1–2 orders of magnitude lower than lysozyme formulations incubated in agitated, siliconized syringes with an air bubble (Fig. 3d). The addition of glass beads to lysozyme formulations incubated in agitated, siliconized syringes with no air bubble increased the number of DOI 10.1002/jps.23973

RESEARCH ARTICLE – Pharmaceutical Biotechnology

particles detected (Fig. 3d; cross symbols) in the solution, but these particle concentrations remained lower than the particle concentrations 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 formulation on a siliconized glass slide is 98◦ ± 1◦ . The interfacial tension of the silicone oil–water interface with 3M adsorbed is ca. 30 mN/m after ca. 40 s (Supplemental Fig. S2). The interfacial 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 under all incubation conditions in PFS. Even protein-free buffer solutions incubated in agitated, siliconized syringes contained a baseline level of particles with a concentration of about 1000 particles/mL. However, roughly three orders of magnitude more particles were generated in protein formulations incubated in agitated, siliconized syringes in the presence of an air bubble. The increase in particle concentrations was seen in both formulations tested. There was a marked difference in the particle concentrations in protein formulations incubated with and without an air bubble in agitated, siliconized syringes. Without an air bubble, the particle concentrations remained between 1000 and 10,000 particles/mL, but the particle concentrations were orders of magnitude higher when an air bubble was present. One potential explanation for the increases in particle concentration in the presence of an air bubble is that bulk fluid shear forces induced by the movement of the air bubble displaced protein aggregates and silicone oil droplets from the syringe wall into the bulk solution. To test this hypothesis, we added glass beads to the syringe, while carefully avoiding the presence of air– water interfaces. As the syringe was rotated, the beads inside the syringe barrel fell, resulting in mixing and bulk fluid shear forces near the silicone oil–water interface but without contributions from the air–water interface to shear. Although it was difficult to quantify their magnitude, we expected that the bulk fluid shear forces exerted by the glass beads (density ca. 2.5) as they passed by the syringe walls were similar to or larger than those exerted by rising air bubbles. Thus, if bulk fluid shear forces and/or mixing were the dominant mechanisms for the production of particles, we would expect that large numbers of particles would be formed when samples were agitated in the presence of glass beads. As seen in Figures 1d and 3d, the addition of glass beads to siliconized syringes did result in higher particle concentrations than those observed in syringes without beads, but the particle concentrations were still significantly lower than those observed in siliconized syringes agitated with an air bubble. Therefore, although bulk fluid shear forces and mixing resulting from air bubble movements may contribute to particle generation they are not the major contributing factor. The effect of bulk fluid shear induced by the motion of the air bubble was also observed in protein formulations incubated in agitated, unsiliconized syringes. Similar to samples that were incubated in siliconized syringes with no air bubble, the particle concentrations in unsiliconized syringes with no air bubble DOI 10.1002/jps.23973

1607

remained less than 10,000 particles/mL. The presence of an air bubble in the unsiliconized syringes increased the particle concentrations but not to the same extent that an air bubble increased the particle concentrations in siliconized syringes. Therefore, some particle generation can be attributed to the bulk fluid shear forces due to the air bubble movement in the syringe. However, the highest particle concentrations observed in this study consistently occurred when the air–water interface and the silicone oil–water interface were both present. It is possible that more particles were generated in siliconized syringes with an air bubble than in unsiliconized syringes with an air bubble because the protein adsorbed to the different interfaces with varying degrees of coverage. The amount of protein required to achieve monolayer surface coverage of 3M adsorbed on glass in 10 mM L-histidine pH 5, measured in this study, is 1.9 ± 0.4 mg/m2 . The amount required to achieve monolayer surface coverage of 3M adsorbed on silicone oil in 10 mM L-histidine pH 5, measured in previous work,3 is 2.7 ± 0.6 mg/m2 . These values are comparable with literature values for proteins adsorbed to solid and fluid interfaces.33,34 Thus, there is only a modest difference in the surface coverage of 3M on siliconized and unsiliconized surfaces, and the substantial difference in particle concentrations between siliconized and unsiliconized syringes with an air bubble must be attributed to other factors. The higher particle concentrations observed in siliconized syringes with an air bubble than in unsiliconized syringes with an air bubble might also be due to silicone oil droplets alone sloughing off the syringe wall into the bulk solution. However, the images of particles captured by FlowCAM clearly showed that the particles did not consist of silicone oil droplets only. In addition to the presence of the silicone oil–water interface and the air–water interface, agitation was also required to yield the highest particle concentrations. In protein formulations incubated in siliconized syringes with no agitation, the particle concentrations remained at the baseline level of 1000– 10,000 particles/mL, and no increase was seen when an air bubble was added. Therefore, movement of the air bubble along the siliconized syringe wall was integral to the generation of particles in PFS. This was consistent with a previous study where the combination of agitation, air–water interfaces, and silicone oil–water interfaces induced more protein aggregation any one stress alone.22 Composition of Particles Detected in PFS It has been suggested that the particles detected in PFS primarily consist of silicone oil droplets and that silicone oil has minimal impact on proteins within the formulations, in particular if the formulation contains surfactant.5,21,23 Droplets of silicone oil characteristically appear in flow microscopy images as spherical shapes.14–16 Images with such spherical shapes were observed under all conditions for formulations incubated in siliconized syringes (Fig. 2 and Supplemental Fig. S3). However, in all of the formulations that contained protein, the images collected during flow microscopy analysis also clearly showed nonspherical particles characteristic of protein aggregates. The most interesting images came from protein formulations agitated in siliconized syringes in the presence of an air bubble (Fig. 2d). In addition to the particle images that appeared to reflect primarily spherically shaped silicone oil droplets or nonspherical protein aggregates, there were images of particles Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

1608

RESEARCH ARTICLE – Pharmaceutical Biotechnology

that consisted of silicone oil droplets apparently coated with aggregated protein. Furthermore, several images showed large agglomerations of protein-coated silicone oil droplets (white highlights in Fig. 2d). These images are similar to the agglomerates of protein aggregates and silicone oil droplets observed in an abatacept formulation incubated in PFS9 and in an IFN$-1a product formulated in PFS.20 Thus, we conclude that the high particle count observed after agitation of protein formulations in siliconized syringes cannot be ascribed solely to the sloughing off of silicone oil droplets; some particles consist of aggregated protein as well. The presence of aggregated protein in a therapeutic product is a major concern because protein aggregates have been shown to elicit an immune response.35–38 Therefore, it is essential to know if the particles detected in a therapeutic product consist of protein aggregates. Several recent studies14–16 have proposed digital filtering techniques based on image analysis to differentiate between particles composed of silicone oil droplets and particles composed of protein aggregates. In each of these studies, pre-formed silicone oil droplets were mixed with pre-formed protein aggregates. These mixtures of silicone oil droplets and protein aggregates were analyzed by microflow imaging, and the resulting images were sorted according to various shape and image intensity criteria, allowing separate populations of silicone oil droplets and protein aggregates to be distinguished and counted. In contrast, the samples in the current study were formed during incubation of protein formulations in siliconized PFS. Particles composed of both silicone oil droplets and aggregated protein can clearly be seen in the microflow images. However, the images also suggest that many particles consist of agglomerates of aggregated protein and silicone oil droplets in addition to particles that are predominantly composed of just silicone oil or just protein. Therefore, current digital filtering techniques are not sufficient to classify the particles detected in this study, and further refinement of these techniques is necessary to accurately categorize particles consisting of both silicone oil and protein. Proposed Mechanism of Particle Generation in Siliconized PFS In this study, the highest particle concentrations in protein formulations in PFS occurred when three elements were all present: (1) silicone oil–water interfaces, (2) air–water interfaces, and (3) agitation. Agitation, in the form of end-over-end rotation, caused the air bubble to move inside the syringe. However, the air bubble movement was not the same in siliconized and unsiliconized syringes (Fig. 4). In unsiliconized syringes, the air bubble moved from one end of the syringe to the other end in less than 1 s when the syringe was rotated. In siliconized syringes, the air bubble moved slowly, requiring about 5 s to move along the syringe wall when the syringe was rotated. Movies of the air bubble movement are included in Supplemental Information. Movement of the air bubble along the siliconized syringe wall is where the three key elements come together, and we hypothesize that this movement is the primary cause of particle generation in siliconized syringes with an air bubble. Proteins readily adsorb to hydrophobic surfaces,26,32,39 such as silicone oil, and are known to form a viscoelastic gel at these surfaces.27–29 When a protein formulation (without surfactant) is filled into a siliconized syringe, the protein immediately begins to adsorb to the siliconized syringe wall. At the protein Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

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

concentration used in this work, the adsorbed protein layer should form a gel within seconds (Fig. 5).40 Because an air bubble is introduced into the syringe during the filling and stoppering process, a three-phase contact line is created where the air–water interface, the silicone oil–water interface, and the silicone oil–air interface meet (Fig. 6). The contact angle at this three-phase contact line is determined by a balance of the interfacial tensions of each of the three interfaces present.41 The value of the interfacial tension at each interface depends on how much protein is adsorbed to that interface; more adsorbed protein decreases the interfacial tension. When the syringe is subjected to agitation (end-over-end rotation in this study), the air bubble moves along the siliconized syringe wall. During this motion, the gelled protein layer at the silicone oil–water interface is disrupted. Capillary forces at the three-phase (silicone oil–water–air) contact line are hypothesized to pull on the gelled protein layer causing the gel to fragment and release gelled protein particles from the silicone oil–water interface into the bulk solution (Fig. 5). In addition, as seen in Figure 2, not only are gelled protein aggregates removed from the interface, but silicone oil is displaced as well because the protein is adsorbed to silicone oil. Therefore, protein aggregates, silicone oil droplets, and agglomerates of protein aggregates and silicone oil droplets are all detected in the bulk formulation. In addition, the particles detected in protein formulations in agitated, siliconized syringes with an air bubble do not change size over time (data not shown). This is consistent with an interfacial mechanism of particle generation where particles are removed from the interface already aggregated rather than a mechanism where protein aggregates are nucleated from an aggregation-prone species and then grow in the bulk. Upon the removal of some of the adsorbed protein from the silicone oil–water interface, the interfacial tension at that interface increases. Thus, the interfacial tension at the silicone oil–water interface at the receding edge of the bubble is expected to be larger than the interfacial tension at the advancing edge of the bubble, where gelled protein is being removed. This is consistent with observations that the advancing contact angle (measured through the liquid) is smaller than the receding contact angle because of the difference in the amount of protein adsorbed at the two edges (Fig. 6). Typically, differences between advancing and receding contact angles are attributed DOI 10.1002/jps.23973

RESEARCH ARTICLE – Pharmaceutical Biotechnology

1609

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 angle between the advancing and receding edges of the bubble cannot be the result of surface roughness. Instead, they are due to a difference in interfacial tension over the length of the bubble, which we ascribe to corresponding differences in the concentrations of adsorbed protein along the interface. Figure 6 shows the balance of the interfacial tensions parallel to the syringe wall at the three-phase contact lines at the advancing and receding edges of the air bubble. This balance of interfacial tensions in the parallel direction is determined by applying the Young equation at the advancing edge of the bubble (indicated by subscript a) and at the receding edge of the bubble (indicated by subscript r):

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

DOI 10.1002/jps.23973

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 ) is 20.9 mN/m.42 At the silicone oil–water and the air–water interfaces, the interfacial tensions will depend on how much protein is adsorbed to those interfaces. The interfacial tensions at the advancing edge of the bubble where protein is adsorbed are estimated based on the values measured by the pendant drop technique after ca. 40 s of adsorption time (corresponding to the time required for one syringe rotation): ca. 65 mN/m for the air–water interfacial tension ((air−liq,a ) and ca. 30 mN/m for the silicone oil–water interfacial tension ((SO−liq,a ). Using these interfacial tension values, the contact angle at the advancing edge (θ a ) is calculated to be 98◦ . An approximate value of this contact angle also can be measured using the contact angle at the three-phase contact line of a 1 :L droplet of 1 mg/mL 3M solution on a siliconized glass slide. This measured angle (measured through the liquid) is 98◦ ± 1◦ , consistent with the calculated value of θ a . The interfacial tensions at the receding edge of the bubble are estimated based on their values when no 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 edge of the bubble (2r, measured through the liquid) is calculated from the Young equation to be 110◦ if all adsorbed protein is removed from that interface. The exact values of the interfacial tensions and the contact angle at the receding edge of the bubble will depend on how much protein is actually removed from that interface, which cannot be measured directly inside the syringe. However, visual observations of the air bubble in the syringe are consistent with a receding contact that is larger than the advancing contact angle. At the three-phase contact line, there is also a component of the air–liquid interfacial tension that is perpendicular to the syringe wall (Fig. 6). Using the approximate diameter of a monoclonal antibody (dprotein = 12 nm), we can estimate the force F perpendicular to the syringe wall acting on one protein molecule at the advancing edge three-phase contact line: F = γ air−liq,a · sin(2a ) · dprotein

(2)

Using the estimated air–water interfacial tension of 65 mN/m and a value θ a = 98◦ , we obtain a value for F of Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

1610

RESEARCH ARTICLE – Pharmaceutical Biotechnology

Figure 6. Schematic of the contact angles at the advancing and receding edges of the air bubble (top), and schematic of the interfacial tensions at the three-phase contact lines at the advancing and receding edges of the air bubble (bottom). The balance of the interfacial tensions parallel to the syringe wall at the advancing and receding edges of the bubble are determined by the Young Equation (Eq. 1a). The interfacial tension and contact angle values are defined in the text. The subscript a corresponds to the advancing edge of the bubble. The subscript r corresponds to the 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 component of the air–liquid interfacial tension that is perpendicular to the syringe wall. The force acting perpendicular to the syringe wall pulling on one protein 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 required to mechanically unfold a protein as measured by atomic force microscopy.43 Therefore, it is likely that a force of this magnitude is strong enough to fragment a gelled protein layer and pull gelled protein particles off the interface. After the air bubble passes over a section of the siliconized syringe wall and removes some of the adsorbed protein, space becomes available on the silicone oil interface for more protein to adsorb from the bulk. This newly adsorbed protein gels, and when the air bubble passes by again, more protein aggregates and silicone oil droplets are removed from the interface and into the bulk. Thus, the particle concentration in siliconized syringes with an air bubble continues to increase with time, as was observed in this study (Figs. 1d and 3d). This interfacial mechanism of particle generation in siliconized syringes is summarized in Figure 5. In unsiliconized syringes, particle generation does not occur in this manner because the air bubble does not move along the syringe wall and interact with the adsorbed protein layer. Instead, the air bubble moves quickly through the center of the syringe. Thus, the gelled protein layer is not disrupted. Interfaces have often been suggested to play a major role in protein aggregation and particle generation in protein

Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

formulations.44–46 However, it is not simply the presence of an interface that causes particle generation. In the PFS used in this study, the silicone oil–water interfacial area available for protein to adsorb is about 10 cm2 /syringe. Based on the surface coverage of the antibody 3M on silicone oil,3 less than 3 :g of 3M can adsorb to this interfacial area. This is a negligible amount of protein compared with the bulk protein concentration in the syringe (1 mg/mL). Furthermore, in the absence of agitation, any protein aggregates that form on the silicone oil– water interface would be expected to stay at the interface and not desorb into the bulk. Single molecule tracking experiments using total internal reflectance fluorescence microscopy show that the surface residence times of protein aggregates on the silicone oil–water interface increase with increasing aggregate size.47,48 However, based on the mechanism proposed above, during agitation, adsorbed protein will be removed from the interface multiple times, and the subsequent replenishment of the adsorbed layer by protein from the bulk will eventually lead to significantly more than just 3 :g of protein interacting with the silicone oil–water interface. Therefore, although the presence of an interface is necessary for a protein gel layer to form, the disruption of the interfacial protein gel layer is the key factor in generating particles. The

DOI 10.1002/jps.23973

RESEARCH ARTICLE – Pharmaceutical Biotechnology

effect of disrupting this layer is apparent in the 1–2 orders of magnitude difference between the particle concentrations in protein formulations with an air bubble in quiescently incubated syringes and in agitated, siliconized syringes (Figs. 1b, 1d, 3b, and 3d; closed symbols). The presence of an air bubble has minimal effect on the particle concentrations when it does not move within the syringe. In addition, there are two orders of magnitude less particles in protein formulations in agitated, siliconized syringes with no air bubble than in agitated, 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 not disrupted, and the particle concentrations do not increase. A disruption of the interfacial protein gel layer has been shown to generate particles in other systems, as well. Tearing and detachment of a gelled protein layer was observed when a Langmuir trough was used to expand and compress the air– water interfacial area.49 In Bee et al.,6 a protein formulation was exposed to an air–water interface that was repeatedly expanded and compressed. Because of the continual expansion and compression of the air–water interface, the gelled protein layer at that interface was disrupted which released particles into the bulk protein solution.6 In another air–water interface study,7 a needle was inserted through the air–water interface of a protein solution. This mechanical disturbance disrupted the gelled protein layer at the air–water interface and released particles into the protein solution. Therefore, we conclude that a major cause of particle generation in protein formulations is a disruption of the protein layer that adsorbs and gels at an interface present in the system. In the current study, the gelled protein layer is disrupted due to the air bubble movement induced by end-over-end rotation of the syringe. However, air bubble movement in the syringe may be induced by a variety of transportation-associated stresses, and it is not necessary that the air bubble move along the entire length of the syringe in order to generate particles.

CONCLUSIONS This study measured the particle concentrations in two different protein formulations after rotation and quiescent incubation in siliconized and unsiliconized glass syringes in the presence and absence of an air bubble. Agitated, siliconized syringes with an air bubble induced the greatest particle generation in both protein formulations tested, and the particles observed in this condition consisted of protein aggregates, silicone oil droplets, and agglomerates of protein aggregates and silicone oil droplets. Although bulk shear forces due to the rotation of the air bubble caused some particle formation, they were not the primary source of particle generation in the agitated, siliconized syringes with an air bubble. Instead, capillary forces at the three-phase contact line caused particle generation and explained the synergistic effects of silicone oil–water interfaces, air–water interfaces, and agitation on particle generation in PFS. Consistent with the mechanism proposed above, there are two options to consider for reducing the number of particles in siliconized syringes with an air bubble. One option is to add a surfactant (such as polysorbate 20) to the protein formulation. Because they are surface active, surfactants can lower the interfacial tension and are commonly used in the pharmaceutical DOI 10.1002/jps.23973

1611

industry to minimize protein adsorption to interfaces.45,50 In addition, surfactants have also been shown to inhibit gelation of adsorbed protein layers.29 The second option involves the silicone oil coating on the syringe wall. The coating should contain the least amount of silicone oil necessary to provide the desired lubrication and should be strongly adhered to the syringe wall to minimize the amount of silicone oil that can slough off. Future experiments are planned to investigate these two options for mitigating the number of particles in protein formulations in PFS.

ACKNOWLEDGMENTS The authors gratefully acknowledge support from MedImmune, Inc. and the National Science Foundation award #CBET1133871.

REFERENCES 1. Jezek J, Darton NJ, Derham BK, Royle N, Simpson I. 2013. Biopharmaceutical formulations for pre-filled delivery devices. Expert Opin Drug Deliv 10:811–828. 2. Fries A. 2009. Drug delivery of sensitive biopharmaceuticals with prefilled syringes. Drug Deliv Technol 9:22–27. 3. Gerhardt A, Bonam K, Bee JS, Carpenter JF, Randolph TW. 2013. Ionic strength affects tertiary structure and aggregation propensity of a monoclonal antibody adsorbed to silicone oil–water interfaces. J Pharm Sci 102:429–440. 4. Li J, Pinnamaneni S, Quan Y, Jaiswal A, Andersson FI, Zhang X. 2012. Mechanistic understanding of protein–silicone oil interactions. Pharm Res 29:1689–1697. 5. Felsovalyi F, Janvier S, Jouffray S, Soukiassian H, Mangiagalli P. 2012. Silicone-oil-based subvisible particles: Their detection, interactions, and regulation in prefilled container closure systems for biopharmaceuticals. J Pharm Sci 101:4569–4583. 6. Bee JS, Schwartz DK, Trabelsi S, Freund E, Stevenson JL, Carpenter JF, Randolph TW. 2012. Production of particles of therapeutic proteins at the air–water interface during compression/dilation cycles. Soft Matter 8:10329–10335. 7. Rudiuk S, Cohen-Tannoudji L, Huille S. 2012. Importance of the dynamics of adsorption and of a transient interfacial stress on the formation of aggregates of IgG antibodies. Soft Matter 8:2651–2661. 8. Badkar A, Wolf A, Bohack L, Kolhe P. 2011. Development of biotechnology products in pre-filled syringes: Technical considerations and approaches. AAPS PharmSciTech 12:564–572. 9. Majumdar S, Ford BM, Mar KD, Sullivan VJ, Ulrich RG, D’Souza AJM. 2011. Evaluation of the effect of syringe surfaces on protein formulations. J Pharm Sci 100:2563–2573. 10. Z¨olls S, Tantipolphan R, Wiggenhorn M, Winter G, Jiskoot W, Friess W, Hawe A. 2012. Particles in therapeutic protein formulations, Part 1: Overview of analytical methods. J Pharm Sci 101:914–935. 11. Singh SK, Afonina N, Awwad M, Bechtold-Peters K, Blue JT, Chou D, Cromwell M, Krause HJ, Mahler HC, Meyer BK, Narhi L, Nesta DP, Spitznagel T. 2010. An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics. J Pharm Sci 99:3302–3321. 12. Carpenter JF, Randolph TW, Jiskoot WIM, Crommelin DJA, Middaugh CR, Winter G, Fan Y, Kirshner S, Verthelyi D, Kozlowski S, Clouse KA, Swann PG, Rosenberg A, Cherney B. 2009. Overlooking subvisible particles in therapeutic protein products: Gaps that may compromise product quality. J Pharm Sci 98:1201–1205. 13. US Pharmacopeia. 2011. Particulate matter in injections. USP–NF, 35 ed. Rockville: USP. 14. Strehl R, Rombach-Riegraf V, Diez M, Egodage K, Bluemel M, Jeschke M, Koulov A. 2012. Discrimination between silicone oil droplets Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

1612

RESEARCH ARTICLE – Pharmaceutical Biotechnology

and protein aggregates in biopharmaceuticals: A novel multiparametric image filter for sub-visible particles in microflow imaging analysis. Pharm Res 29:594–602. 15. Weinbuch D, Z¨olls S, Wiggenhorn M, Friess W, Winter G, Jiskoot W, Hawe A. 2013. Micro – flow imaging and resonant mass measurement (Archimedes)—Complementary methods to quantitatively differentiate protein particles and silicone oil droplets. J Pharm Sci 102:2152– 2165. 16. Z¨olls S, Weinbuch D, Wiggenhorn M, Winter G, Friess W, Jiskoot W, Hawe A. 2013. Flow imaging microscopy for protein particle analysis— A comparative evaluation of four different analytical instruments. AAPS J 15:1200–1211. 17. Joubert MK, Luo Q, Nashed-Samuel Y, Wypych J, Narhi LO. 2011. Classification and characterization of therapeutic antibody aggregates. J Biol Chem 286:25118–33. 18. Simler B, Hui G. 2012. Mechanistic complexity of subvisible particle formation: Links to protein aggregation are highly specific. J Pharm Sci 101:4140–4154. 19. Liu L, Ammar DA, Ross LA, Mandava N, Kahook MY, Carpenter JF. 2011. Silicone oil microdroplets and protein aggregates in repackaged bevacizumab and ranibizumab: Effects of long-term storage and product mishandling. Invest Ophthalmol Vis Sci 52:1023–1034. 20. Barnard J, Babcock K, Carpenter J. 2013. Characterization and quantitation of aggregates and particles in interferon-$ products: Potential links between product quality attributes and immunogenicity. J Pharm Sci 102:915–928. 21. Lubiniecki A, Volkin DB, Federici M, Bond MD, Nedved ML, Hendricks L, Mehndiratta P, Bruner M, Burman S, Dalmonte P, Kline J, Ni A, Panek ME, Pikounis B, Powers G, Vafa O, Siegel R. 2011. Comparability assessments of process and product changes made during development of two different monoclonal antibodies. Biologicals 39:9– 22. 22. Thirumangalathu R, Krishnan S, Ricci MS, Brems DN, Randolph TW, Carpenter JF. 2009. Silicone oil- and agitation-induced aggregation of a monoclonal antibody in aqueous solution. J Pharm Sci 98:3167–3181. 23. Auge K, Blake-Haskins A, Devine S, Rizvi S, Li Y, Hesselberg M, Orvisky E, Affleck R, Spitznagel T, Perkins M. 2011. Demonstrating the stability of albinterferon alfa-2b in the presence of silicone oil. J Pharm Sci 100:5100–5114. 24. Basu P, Krishnan S, Thirumangalathu R, Randolph TW, Carpenter JF. 2013. IgG1 aggregation and particle formation induced by silicone– water interfaces on siliconized borosilicate glass beads: A model for siliconized primary containers. J Pharm Sci 102:852–865. 25. Ludwig DB, Carpenter JF, Hamel J-B, Randolph TW. 2010. Protein adsorption and excipient effects on kinetic stability of silicone oil emulsions. J Pharm Sci 99:1721–1733. 26. Dixit N, Maloney KM, Kalonia DS. 2011. Application of quartz crystal microbalance to study the impact of pH and ionic strength on protein–silicone oil interactions. Int J Pharm 412:20–27. 27. Petkov J, Gurkov T, Campbell B, Borwankar R. 2000. Dilatational and shear elasticity of gel-like protein layers on air/water interface. Langmuir 16:3703–3711. 28. Bantchev G, Schwartz D. 2003. Surface shear rheology of betacasein layers at the air/solution interface: Formation of a twodimensional physical gel. Langmuir 19:2673–2682. 29. Liu L, Qi W, Schwartz D, Randolph TW, Carpenter JF. 2013. The effects of excipients on protein aggregation during agitation: An interfacial shear rheology study. J Pharm Sci 102:2460–2470. 30. Oganesyan V, Damschroder MM, Leach W, Wu H, Dall’Acqua WF. 2008. Structural characterization of a mutated, ADCC-enhanced human Fc fragment. Mol Immunol 45:1872–1882.

Gerhardt et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1601–1612, 2014

31. Bee JS, Chiu D, Sawicki S, Stevenson JL, Chatterjee K, Freund E, Carpenter JF, Randolph TW. 2009. Monoclonal antibody interactions with micro- and nanoparticles: Adsorption, aggregation, and accelerated stress studies. J Pharm Sci 98:3218–3238. 32. Beverung CJ, Radke CJ, Blanch HW. 1999. Protein adsorption at the oil/water interface: Characterization of adsorption kinetics by dynamic interfacial tension measurements. Biophys Chem 81:59–80. 33. Nakanishi K, Sakiyama T, Imamura K. 2001. On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon. J Biosci Bioeng 91:233–244. 34. McClements DJ. 2005. Food emulsions: Principles, practices, and techniques, 2nd ed. Boca Raton: CRC Press. 35. Biro CE, Garcia G. 1965. The antigenicity of aggregated and aggregate-free human gamma-globulin for rabbits. Immunology 8:411– 419. 36. Braun A, Kwee L, Labow M, Alsenz J. 1997. Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha in normal and transgenic mice. Pharm Res 14:1472–1478. 37. Rosenberg AS. 2006. Effects of protein aggregates: An immunologic perspective. AAPS J 8:E501–E507. 38. Fradkin A, Carpenter J, Randolph T. 2009. Immunogenicity of aggregates of recombinant human growth hormone in mouse models. J Pharm Sci 98:3247–3264. 39. Dickinson E. 1999. Adsorbed protein layers at fluid interfaces: Interactions, structure and surface rheology. Colloids Surf 15:161– 176. 40. Mehta S, Lewus R, Bee JS, Randolph TW, Carpenter JF. Unpublished results. Gelation of a monoclonal antibody at silicone oil–water interfaces and its subsequent rupture results in aggregation. 41. Adamson AW. 1990. Physical chemistry of surfaces, 5th ed. New York: John Wiley and Sons, Inc. 42. Nakamura K, Refojo M, Crabtree D. 1990. Factors contributing to the emulsification of intraocular silicone and fluorosilicone oils. Invest Ophthalmol Vis Sci 31:647–656. 43. Brockwell DJ, Beddard GS, Paci E, West DK, Olmsted PD, Smith DA, Radford SE. 2005. Mechanically unfolding the small, topologically simple protein L. Biophys J 89:506–19. 44. Mahler H, Fischer S, Randolph T, Carpenter J. 2010. Protein aggregation and particle formation: Effects of formulation, interfaces, and drug product manufacturing operations. In Aggregation of therapeutic proteins; Wang W, Roberts C, Eds. Hoboken: John Wiley and Sons Ltd. 45. Bee JS, Randolph TW, Carpenter JF, Bishop SM, Dimitrova MN. 2011. Effects of surfaces and leachables on the stability of biopharmaceuticals. J Pharm Sci 100:4158–4170. 46. Pinholt C, Hartvig RA, Medlicott NJ, Jorgensen L. 2011. The importance of interfaces in protein drug delivery—Why is protein adsorption of interest in pharmaceutical formulations? Expert Opin Drug Deliv 8:949–964. 47. Walder R, Schwartz DK. 2010. Single molecule observations of multiple protein populations at the oil–water interface. Langmuir 26:13364–13367. 48. Walder R, Schwartz DK. 2011. Dynamics of protein aggregation at the oil–water interface characterized by single molecule TIRF microscopy. Soft Matter 7:7616–7622. 49. Van Aken GA, Merks MTE. 1996. Adsorption of soluble proteins to dilating surfaces. Colloids Surf A Physicochem Eng Asp 114:221– 226. 50. Lee HJ, McAuley A, Schilke KF, McGuire J. 2011. Molecular origins of surfactant-mediated stabilization of protein drugs. Adv Drug Deliv Rev 63:1160–1171.

DOI 10.1002/jps.23973