IMM61
`Silicone Oil Microdroplets and Protein Aggregates in
`Repackaged Bevacizumab and Ranibizumab: Effects of
`Long-term Storage and Product Mishandling
`
`Lu Liu, 1 David A. Ammar, 2 Lindsey A. Ross, 3 Naresh Mandava, 2 Malik Y. Kahook, 2
`and John F. Carpenter1
`
`Puru>osE. To quantify levels of subvisible particles and protein
`aggregates in repackaged bevacizumab obtained from com(cid:173)
`pounding pharmacies, as well as in samples of bevacizumab
`and ranibizumab tested in controlled laboratory experiments.
`METHODS. Repackaged bevacizumab was purchased from four
`external compounding pharmacies. For controlled laboratory
`studies, bevacizumab and placebo were drawn into plastic
`syringes and incubated at -20°C, 4°C, and room temperature
`(with and without exposure to light) for 12 weeks. In addition,
`mechanical shock occurring during shipping was mimicked
`with syringes containing bevacizumab. Particle counts and size
`distributions were quantified by particle characterization tech(cid:173)
`nology. Levels of monomer and soluble aggregates of bevaci(cid:173)
`zumab were determined with size-exclusion high-performance
`liquid chromatography (SE-HPLC) .
`REsmTs. Repackaged bevacizumab from the compounding
`pharmacies had a wide range of particle counts (89,006 ±:
`56,406 to 602 ,062 ±: 18,349/mL). Bevacizumab sampled di(cid:173)
`rectly from the original glass vial had particle counts of
`63,839 ±: 349/mL. There was up to a 10% monomer loss in the
`repackaged bevacizumab. Laboratory samples of repackaged
`bevacizumab and placebo had initial particle counts, respec(cid:173)
`tively, of 283,675 ±: 60,494/mL and 492,314 ±: 389,361/mL.
`Freeze-thawing of both bevacizumab and placebo samples led
`to > 1.2 million particles/mL. In all repackaged samples, most
`of the particles were due to silicone oil. SE-HPLC showed no
`significant differences for repackaged samples incubated in the
`laboratory under various conditions, compared with bevaci(cid:173)
`zumab directly from vial. However, repeated freeze-thawing
`caused a more than 10% monomer loss.
`CONCLUSIONS. Bevacizumab repackaged in plastic syringes
`could contain protein aggregates and is contaminated by
`silicone oil microdroplets. Freeze-thawing or other mishan(cid:173)
`dling can further increase levels of particle contaminants.
`
`From the 1Department of Pharmaceutical Sciences, Center for
`Pharmaceutical Biotechnology and the 3School of Pharmacy, University
`of Colorado Denver, Aurora, Colorado; and the 2 Rocky Mountain Lions
`Eye Institute, Department of Ophthalmology, University of Colorado
`School of Medicine, Aurora, Colorado.
`Supported by National Institutes of Health Grant 5R01-EB006006.
`Submitted for publication August 18, 2010; revised September 17,
`2010; accepted September 27, 2010.
`Disclosure: L. Liu, None; D.A. Ammar, None; L.A. Ross, None;
`N. Mandava, Genentech (F); M.Y. Kahook, Genentech (C); J.F.
`Carpenter, BD Biosciences (F, R)
`Corresponding author: John F. Carpenter, University of Colorado
`Denver School of Pharmacy, Department of Pharmaceutical Sciences,
`12700 E. 19th Avenue, Mail Stop C-238, Aurora, CO 80045;
`john.carpenter@ucdenver.edu.
`
`Investigative Ophthalmology & Visual Science, February 2011, Vol. 52, No. 2
`Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
`
`(Invest Ophthalmol Vis Sci. 2011;52:1023-1034) DOI:
`10.1167 /iovs.10-6431
`
`B evacizumab (Avastin; Genentech Technology, Inc., South
`
`San Francisco, CA) is a recombinant human monoclonal
`antibody that inhibits endothelial cell growth and subsequent
`vascularization. It was approved for intravenous (IV) treatment
`of metastatic colorectal cancer by the United States Food and
`Drug Administration (FDA) in 2004 and subsequently has been
`approved for IV treatment of non-small cell lung cancer, met(cid:173)
`astatic breast cancer, glioblastoma, and metastatic kidney can(cid:173)
`cer. Because of its antivascular activity, bevacizumab has also
`been used by ophthalmologists for the off-label treatment of
`wet age-related macular degeneration (AMD). This practice has
`grown rapidly because repackaged bevacizumab has been very
`effective in treating AMD 1 and because the cost per dose of
`bevacizumab is substantially lower than that of ranibizumab
`(Lucentis; Genentech), which is an FDA approved anti-VEGF
`agent specifically packaged and sold for the treatment of wet
`AMD. 1
`The apparent safety and efficacy of intravitreal bevacizumab
`have been supported by published peer-reviewed reports2
`5
`-
`and have led the National Eye Institute (NEI) to undertake a
`large randomized double-masked multicenter clinical trial
`(Comparison of AMD Treatments Trials [CATT]) to compare
`bevacizumab with ranibizumab. 6 However, reports of sus(cid:173)
`tained elevation of intraocular pressure (IOP) and inflammation
`after the intravitreal use of bevacizumab and ranibizumab have
`been increasing. 7
`13 Recently, we proposed that some in(cid:173)
`-
`creases in IOP could be due to particulate matter present in
`bevacizumab, which for off-label use is typically repackaged in
`plastic syringes. 14 In support of this hypothesis, our investiga(cid:173)
`tion documented that there were both protein aggregates and
`particles ::o- 1 µm in repackaged bevacizumab obtained from
`three external compounding pharmacies.
`Many factors (e .g., storage time, type of syringe, freeze(cid:173)
`thawing, and mechanical shock during shipping) that could
`affect particle and protein aggregate levels and sizes in repack(cid:173)
`aged bevacizumab have not been investigated. Furthermore,
`determining the ranges of values for these critical product
`characteristics in samples purchased from different external
`compounding pharmacies is important for the field. In the
`present study, we addressed these problems with repackaged
`bevacizumab. First, we quantified the levels of subvisible par(cid:173)
`ticles and protein aggregates in bevacizumab in plastic syringes
`purchased from four external compounding pharmacies. Sec(cid:173)
`ond, we performed controlled laboratory experiments with
`bevacizumab repackaged in the same types of plastic syringes
`as those used by the external compounding pharmacies. We
`tested the effects of storage at room temperature (with and
`without exposure to light), -20°C, and 4°C on particle counts
`and protein aggregates and the effects of repetitive freeze-
`
`1023
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`
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`

`1024 Liu et al.
`
`IOVS, February 2011, Vol. 52, No. 2
`
`thawing. In addition, we studied the potential for freeze-thaw(cid:173)
`ing of samples in shipping containers used by the external
`compounding pharmacies and the effects of mechanical shock
`due to handling mimicking that occurring during shipping.
`We used a particle characterization technique (MicroFlow
`Imaging [MFI]; Brightwell Technologies, Ottawa, ON, Canada)
`to count and size particles ::o, 1 µm and size-exclusion high(cid:173)
`performance liquid chromatography (SE-HPLC) to quantify lev(cid:173)
`els of monomeric and aggregated bevacizumab. Also, we con(cid:173)
`ducted analyses and experiments that documented that most
`of the particles were due to silicone oil microdroplets. Finally,
`for comparison to results with repackaged bevacizumab, we
`also quantified the subvisible particles present in ranibizumab
`samples.
`In this article, we report that bevacizumab repackaged in
`plastic syringes was contaminated by silicone oil microdrop(cid:173)
`lets. Freeze-thawing and mechanical shock can further degrade
`the product and increase levels of particle contaminants. Al(cid:173)
`though we cannot directly link silicone oil contaminants and
`bevacizumab aggregates with sustained elevation of IOP and
`inflammation at this time, based on current studies, mishan(cid:173)
`dling of repackaged bevacizumab should be and can be
`avoided.
`
`MATERIALS AND METHODS
`
`Bevacizumab was purchased from the University of Colorado Hospital
`Inpatient Pharmacy. It was supplied as 25 mg/mL X 4 mL in preser(cid:173)
`vative-free, single-use glass vials. Unless otherwise indicated, for the
`controlled laboratory studies, 0.3-mL insulin syringes (BD Ultra-Fine
`Short; Cat. No. 08290-328438; BD Biosciences, Franklin Lakes, NJ)
`were used. All chemicals used were of reagent grade or higher.
`Repackaged bevacizumab in insulin plastic syringes was purchased
`from four external compounding pharmacies ( designated CP l -CP4)
`on two different occasions. For each compounding pharmacy, the
`details of the syringe configurations and the shipping materials used
`are described in the Results section.
`Ranibizumab was obtained from the residual solution remaining in
`the vials after clinical administration at the University of Colorado
`Hospital Eye Center. The residual solution was collected on the day of
`administration, stored at 4°c in the dark, and used in laboratory
`experiments within 24 hours.
`
`Observations on Shipping Containers Used for
`Repackaged Bevacizumab
`On arrival of the repackaged bevacizumab from each of the four
`compounding pharmacies, observations were made of the inside and
`outside of each shipping box; the orientation, weight, and surface
`temperature of the gel packs; the wrapping materials in which the
`syringes were placed; the relative position of syringes, gel packs, and
`wrapping materials; and the type of syringe. A thermocouple (Dual
`thermometer; Fisher Scientific, Pittsburgh, PA) was attached to the
`surface of a gel pack to determine the temperature on arrival.
`
`Testing of the Potential for the Formulation to
`Freeze in Syringes in Shipping Containers
`First, the gel packs obtained from the respective shipping containers
`(Styrofoam boxes) used by CPl and CP3 were placed in a -20° freezer
`overnight. Then, the gel packs were placed in the respective Styrofoam
`shipping boxes, in the orientation observed on arrival of the packages.
`A thin wire thermocouple was placed on the surface of a gel pack in
`each box, the box was closed with the lid, and the temperature on the
`surface of the gel pack was recorded for 45 hours.
`To test the potential for repackaged bevacizumab to freeze during
`shipping, we inserted a thin wire thermocouple into 0.05 mL of
`placebo solution (60 mg/mL trehalose · 2H2 0, 5.8 mg/mL NaH2P0 4 •
`H2 0, 1.2 mg/mL Na2 HP04 , and 0.4 mg/mL polysorbate 20; pH6.2)
`
`contained in 0.3-mL insulin syringes. The syringes were in direct
`contact with the surface of gel packs that had just been removed from
`the - 20°c freezer. The gel packs and syringes were placed in the
`Styrofoam shipping boxes that were obtained from CPl and CP3. The
`temperature of the placebo in the syringes was recorded with the dual
`digital thermometer as a function of time. In another experiment, to
`avoid potential nucleation of ice by the thermocouple immersed in the
`solution inside the syringes, we attached the thermocouple to the outside
`surface of the syringes at the level of the solution inside. The syringes were
`placed in contact with the gel packs in the Styrofoam shipping containers,
`as just described, and the temperature was recorded as a function of time.
`At various times, the syringes were taken out of the box for visual
`inspection to see whether the placebo had frozen. We also tested the
`temperature on the surface of the syringes (containing 0.05 mL placebo),
`which were wrapped with additional multilayers of bubble packing ma(cid:173)
`terial and placed horizontally or vertically between the gel packs.
`
`Analytical Methods for Particles and Protein
`Monomer and Aggregates
`[MFI];
`Particle characterization
`technology (MicroFlow Imaging
`DPA4100 Flow Microscope, Series B; Brightwell Technologies) was
`used to capture digital images of particles, quantify particle counts, and
`determine size distributions (:c>: 1 µm). The flow cell (100 µm) was used
`in SP3 mode after calibration with a 10-µm polystyrene microsphere
`standard (cat. no. 4210A; Duke Scientific Corp., Palo Alto, CA). Ultra(cid:173)
`pure water (0.22 µm filtered; Millipore, Billerica, MA) was used to
`check the background counts during MFI. The total volume of sample
`dispensed into the flow cell was 0.3 mL, and 0.15 mL was run into the
`cell before the start of data acquisition. The particle count for placebo
`was subtracted from that of the bevacizumab samples. Then, if appli(cid:173)
`cable, to obtain the particle counts before sample dilution, the particle
`counts were multiplied by the dilution factor (see below) used in
`preparation of bevacizumab samples for analysis.
`SE-HPLC was used to fractionate and quantify monomer and soluble
`aggregates in the bevacizumab samples. The samples were centrifuged
`(10 minutes at 14,000g) to pellet any insoluble material (potentially
`including protein precipitates). The supernatants were analyzed by
`using the SE-HPLC mode on an asymmetric flow field flow fractionation
`system (Eclipse AFFF; Wyatt Technology Corp., Santa Barbara, CA) that
`connected with a multiangle light-scattering detector (Dawn EOS;
`Wyatt Technology Corp.) and a UV detector (Gold Module 166; Beck(cid:173)
`man Coulter, Fullerton, CA). The mobile phase (0.182 M KH2P04 ,
`0.018 M K2HP0 4 , and 0.25 M KC! [pH 6.2]) was run at a flow rate of
`0.5 mL/min through a gel column (TSK-GEL G3000SWxl; Tosoh Bio(cid:173)
`science, Tokyo, Japan), and elution was monitored at 280 nm. The
`volume of injection was 100 µL, and the running time was 30 minutes.
`The sum of the peak areas (monomer and the earlier eluting oligomer)
`of the chromatograph for samples directly from the glass vial was
`calculated (ASTRA V software; Wyatt Technology Corp.) and served as
`the control value for the repackaged samples. The percentage of
`monomer and the earlier-eluting oligomer in repackaged samples was
`obtained via dividing the respective peak areas by the summed peak
`area for the control sample and multiplying by 100.
`
`Effects of Preanalysis Dilution on Particle Counts
`
`The volume of repackaged bevacizumab obtained from compounding
`pharmacies was either 0.05 or 0.1 mL, and 0.05 mL was used in our
`controlled laboratory studies in insulin syringes, as described in the
`next section. Analysis by MFI requires at least 0.3-mL samples to give
`accurate particle counts. To be able to obtain data for samples from
`individual syringes, it was necessary to dilute the bevacizumab formu(cid:173)
`lation to a sufficient volume so that the assay could be run properly.
`Dilutions were made in the placebo. To ascertain whether dilution
`changed the particle number and size distribution, MFI analysis was
`performed on undiluted bevacizumab formulation (from repackaged
`syringes) and samples diluted 1:10, 1:20, 1:30, 1:40, and 1:50 with
`placebo.
`
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`Regeneron Exhibit 1025.002
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`

`IOVS, February 2011, Vol. 52, No. 2
`
`Effects of Product Mishandling on Repackaged Bevacizumab
`
`1025
`
`Analysis of Repackaged Bevacizumab from
`Compounding Pharmacies
`
`Particle counts and size distributions (2- 1 µm) of the repackaged
`bevacizumab samples were determined by MFI after 1 :30 dilution with
`placebo. Monomer and soluble aggregates in the diluted bevacizumab
`samples were measured by SE-HPLC. The solution in the syringe was
`expelled by pushing the plunger, and the sample was collected in a
`2-mL cryogenic tube and then diluted with the appropriate volume of
`placebo. Shipments of syringes with repackaged bevacizumab were
`obtained on two separate occasions. For the first shipment, the sam(cid:173)
`ples were analyzed on receipt. For the second shipment, a set of
`syringes from each compounding pharmacy were analyzed immedi(cid:173)
`ately on receipt. Another set was stored at 4°c to within 14 days of the
`3-month expiration date, which was used by all the external com(cid:173)
`pounding pharmacies for repackaged bevacizumab.
`
`Controlled Laboratory Studies of
`Repackaged Bevacizumab
`Sample Preparation. In a laminar flow hood, bevacizumab was
`drawn (0.05 mL, unless otherwise noted) into the 0.3-mL insulin
`syringes (cat. no. 328438; BD Biosciences) through the rubber closure
`of the vial. The 0.3-mL insulin syringes were used in all experiments
`unless otherwise indicated. For comparison, bevacizumab placebo (60
`mg/mL trehalose · 2H2 0, 5.8 mg/mL NaH2P0 4
`• H20, 1.2 mg/mL
`Na2HP0 4 , and 0.4 mg/mL polysorbate 20 [pH 6.2]) was prepared in the
`same type of syringe.
`Three-Month Incubation Study. Syringes with 0.05 mL of
`bevacizumab were incubated at room temperature (with and without
`exposure to laboratory fluorescent lights), -20°c, and 4°c for 0, 1, 2,
`4, 8, and 12 weeks. Particle counts and size distribution (2- 1 µm) in the
`incubated bevacizumab samples were determined by MFI. Monomer
`and soluble aggregates in the bevacizumab samples were measured by
`SE-HPLC.
`Repeated Freeze-thawing. Samples of 0.05 mL bevacizumab
`in syringes were frozen at -20°c and thawed at 4°c, 1, 5, and 10
`times. MFI and SE-HPLC were used to characterize the freeze-thaw
`samples.
`Effect of Syringe Type during Freeze-thawing. To com(cid:173)
`pare 1.0-mL syringes with 0.3-mL syringes, we drew the bevacizumab
`into 1.0-mL tuberculin syringes (cat. no. 309602; BD Biosciences)
`through a dispensing pin (Mini-Spike Dispensing Pin, product code:
`DPlOOOSC; B. Braun Medical, Inc., Bethlehem, PA) which entered the
`rubber closure of the vial. The samples syringes were placed in a
`-20°C freezer and incubated for 2 weeks, and the solution was thawed
`at 4°C. MFI and SE-HPLC were used to characterize the freeze-thaw
`samples.
`Mechanical Shock of Syringes Containing Repackaged
`Bevacizumab. Syringes containing 0.05 mL bevacizumab were
`placed between unfrozen gel packs in one of the Styrofoam shipping
`boxes obtained from CP3. To mimic the mechanical shock to which
`the samples could be subjected during shipping, the box was tossed
`horizontally at a height of 1.2 m and for a distance of approximately
`2 m. This process was repeated 20 times. MFI and SE-HPLC were used
`to characterize the samples after the tossing.
`Characterization of Particles Measured with MFI. To
`gain insight into the identity of the component material of these
`particles, we conducted experiments to confirm that most of the
`particles counted by MFI in the bevacizumab samples from plastic
`syringes were silicone oil. First, we prepared samples in syringes and
`incubated them for 3 weeks at 4°c and -20'C. We prepared a suffi(cid:173)
`cient number of syringes so that, after incubation, pooling of the
`expelled bevacizumab provided enough sample volume that analysis
`could be performed without dilution. This method allowed us to
`observe in images from the MFI the full population of particles that
`were present without dilution (see the Results section, Fig. 6, for
`representative images) and to observe many more particles per image
`
`than noted after dilution. In addition, samples of bevacizumab or
`placebo were freeze-thawed in the plastic syringes and the results
`compared to those of the samples of placebo freeze-thawed in cryo(cid:173)
`genic vials, which are free of silicone oil. We directly drew 0.3 mL of
`bevacizumab or placebo into 0.3-mL insulin syringes. Placebo (0.3 mL)
`was pipetted into 2 mL cryogenic vials. Also, we prepared a suspension
`of microdroplets of silicone oil (160 µg/mL; 1000 cSt; Dow Coming,
`Midland, MI) in placebo with a vortex mixer (Vortex Genie2, cat. no.
`12-812, Thermo Fisher Scientific, Inc., Pittsburgh, PA). The suspension
`of silicone oil microdroplets (0.3 mL) was pipetted into cryogenic vials.
`To confirm that the spherical objects in the images were not air
`bubbles, we tested the effects of degassing. Samples (bevacizumab or
`placebo in plastic syringes, placebo in cryogenic vials, and placebo
`spiked with silicone oil in cryogenic vials) were frozen at -20°C and
`thawed at 4°C. For comparison, another set of samples was kept at
`room temperature. After that, each of the sample types, either with or
`without freeze-thawing, was degassed under vacuum (20 in. of mer(cid:173)
`cury, i.e., 20 in. HgV) for 3 hours to remove any potential micro(cid:173)
`bubbles of air before MFI analysis. For comparison, another set of each
`of the sample types was kept at room temperature without degassing.
`These studies allowed comparison of particles found in placebo sam(cid:173)
`ples taken from plastic syringes and cryogenic vials, as well as in an
`authentic silicone oil sample, with the particles found in the repack(cid:173)
`aged bevacizumab taken from plastic syringes.
`Particle Counts of Ranibizumab. The particle counts and
`size distributions of the ranibizumab samples were obtained by MFI for
`the following preparations: removed directly from the glass product
`vials with a plastic tip on a pipetter; drawn without filtration from the
`vial into a 1-mL tuberculin syringe and then expelled for measurement;
`and according to preparation for administration as described in the
`ranibizumab prescribing information. In this case, the ranibizumab
`formulation was withdrawn from the vial through a filter needle
`(Nokor filter needle with 5-µm wall, 19-gauge X 1.5 in., reference No.
`305200; BD Biosciences) attached to a 1-mL tuberculin syringe, and
`then, after removal of the filter, the solution was expelled for measure(cid:173)
`ment. For all methods of sample handling, after the sample was ob(cid:173)
`tained, the ranibizumab was diluted 1:30 with ranibizumab placebo
`(10 mM histidine HCl, 10% a-trehalose dehydrate, and ().()1% polysor(cid:173)
`bate 20 [pH 5.5]), and then the particles were counted.
`
`RESULTS
`
`Observations on Shipping Containers Used for
`Repackaged Bevacizumab
`The shipping systems for the repackaged bevacizumab varied
`among the compounding pharmacies (fable 1). In particular,
`the weight and orientation of the gel packs used as cold media,
`and the relative position of individual ~yringes to the gel packs
`varied greatly (fable 1). The materials wrapped around the
`syringes of the repackaged bevacizumab were also different. In
`some cases (CPI, -2, and -4), the sytinges were first packed
`individually in a tr-.msparent plastic bag, which was then placed
`in a large brown or silver plastic ziplock bag. In one case (CP3),
`the syringes were initially packed individually in small black
`plastic bags, which were then placed inside a large transparent
`ziplock plastic bag. Samples from CP3 had an additional layer
`of bubble wrap packing materials outside the large ziplock bag.
`On arrival, after overnight shipping, the surface temperature of
`the gel packs from all four compounding pharmacies was 1 °C.
`The plastic syringes used by the four compounding phar(cid:173)
`macies to repackage bevacizumab were also different (Table
`1). CP2, CP3, and CP4 used 0.3-mL insulin ~yringes with perma(cid:173)
`nently attached needles. The needle lengths were 'Yic, in. (8 mm)
`on ~yringes obtained from CP3 and CP4 and Y2 inch (12.7 mm) on
`those obtained from CP2. Only one compounding pharmacy
`(CPI) used 1-mL tuberculin sytinges with a Luer-Slip fitting to
`which a new needle must be attached before intr-,ivitreal injec-
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`1026 Liu et al.
`
`IOVS, February 2011, Vol. 52, No. 2
`
`TABLE 1. Details of the Repackaged Bevacizumab
`
`Compounding Total Weight of Orientation of
`Pharmacy
`Gel Packs (kg)
`Gel Packs
`
`Relative Position of
`Repackaged Bevacizumab
`
`Syringe Reference
`Number
`
`CPl
`CP2
`CP3
`CP4
`
`0.34
`1.14
`0.79
`0.57
`
`Vertical
`Vertical
`Horizontal
`Horizontal
`
`Between two gel packs
`Between three gel packs
`Between two gel packs
`Under two gel packs
`
`309602
`328440
`328438
`309301
`
`Data show the weight and orientation of gel packs, the relative position of repackaged bevacizumab
`to gel packs, and the reference number of plastic syringes used by the four external compounding
`pharmacies to repackage bevacizumab.
`
`tion. A black rubber cap on the Luer-Slip fitting was used to seal
`the 1-mL ~yringe.
`
`Testing of Potential for Formulation to Freeze in
`Syringes in Shipping Containers
`After the gel packs equilibrated in the -20°C freezer, they
`were taken out and placed into their original shipping boxes.
`The surface temperatures of the gel packs remained below 0°C
`for up to 5 hours (Fig. IA). Then, the temperature remained at
`1 °C for 20 to 40 hours, depending on the weight and initial
`
`(.)
`
`30
`
`10
`
`- 20
`0 -<I>
`1-::, - 0
`
`C'CS
`1-
`
`<I> a. -10
`E
`<I>
`-20
`I-
`-30
`
`0
`
`V
`
`CP3
`CP1
`
`A
`
`0 5 10 15 20 25 30 35 40 45
`
`Time(Hour)
`
`0
`
`V
`
`CP3
`CP1
`
`B
`
`- 20
`0 -<I>
`1-::, - 0 ~~ C'CS
`
`temperature of the gel packs when they were placed into the
`shipping boxes. The temperature of the placebo in the syringes
`in contact with the gel packs-removed from the - 20°C
`freezer and placed in the shipping container- cooled below
`0°C, and the solution froze in less than 10 minutes (Fig. IB) . To
`avoid potential nucleation of placebo due to the thermocouple
`directly contacting the placebo solution, the thermocouple
`was attached to the outside surface of syringes. The sample
`temperatures dropped as low as -17°C (data not shown) and
`the samples froze within 30 minutes. Also, we tested placebo(cid:173)
`containing syringes wrapped with an additional multilayer of
`bubble packing material. In syringes placed horizontally be(cid:173)
`tween the gel packs (CP3; Table 1), the placebo solution froze
`within 30 minutes. The temperature of samples placed verti(cid:173)
`cally between the gel packs (CPI; Table 1) dropped to -I2°C,
`but the solution did not freeze within 30 minutes ( data not
`shown). Clearly, at this degree of supercooling, freezing would
`be expected to occur ultimately during actual shipping. The
`results presented in Figure 1 demonstrate that there is potential
`for repackaged bevacizumab to undergo freeze-thawing during
`the shipping process. However, unless temperature monitor(cid:173)
`ing equipment is used during actual shipping from compound(cid:173)
`ing pharmacies, the temperature profiles during the shipping
`of repackaged bevacizumab cannot be assessed.
`
`Effect of Preanalysis Dilution on Particle Counts
`
`Compared with undiluted bevacizumab formulation, the parti(cid:173)
`cle number and size distribution of samples diluted 1:30, 1:40,
`and 1:50 with placebo did not show substantial differences
`from values obtained without dilution (data not shown). In
`contrast, the particle number and size distribution of samples
`diluted 1:10 and 1:20 did differ from the values obtained
`without dilution, but due to the variability of the results for
`individual replicates, the differences were not statistically sig(cid:173)
`nificant (P > 0.05) when comparing the mean particle counts
`for each dilution to that for the undiluted sample by Student's
`t-test. We chose a 1:30 dilution for all repackaged bevacizumab
`samples, unless otherwise indicated.
`
`Analysis of Repackaged Bevacizumab from
`Compounding Pharmacies
`
`The repackaged bevacizumab obtained in the first batch from
`the compounding pharmacies (no less than duplicate samples
`tested, except for CP2) had a wide range of particle counts
`(89,006 ±: 56,406 /mL to 602,062 ±: 18,349 /mL; Fig. 2A). For
`comparison, bevacizumab sampled directly from the original
`glass vials had a particle count of 63,839 ±: 349/mL. Particle
`counts also varied greatly in the same batch of repackaged
`bevacizumab from CP3 (Fig. 3). Representative particle images
`of the CP3 and CP4 samples (Fig. 2) showed that there were
`some large size particles (e.g., 111 µm) in the CP3 samples and
`various sizes of dark spherical particles in the CP4 samples that
`were later confirmed to be silicone oil microdroplets.
`
`(.)
`
`30
`
`10
`
`1-
`
`<I> a. -10
`E
`<I>
`-20
`I-
`-30
`
`0
`
`5
`
`10 15 20 25 30 35
`
`Time(Minute)
`
`FIGURE 1. The temperatures of gel packs (A) and placebo in the
`syringe (B) as a function of time after the gel packs from CPl and CP3
`were kept in a - 20°c freezer overnight and placed into their original
`shipping boxes. (A) Thermocouples were directly attached to the
`surface of the gel packs; (B) thermocouples were inserted into 50 µL
`of placebo solution in the syringes which directly contacted the sur(cid:173)
`face of the gel packs.
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932965/ on 10/09/2017
`
`Regeneron Exhibit 1025.004
`
`

`

`IOVS, February 2011, Vol. 52, No. 2
`
`Effects of Product Mishandling on Repackaged Bevacizumab
`
`1027
`
`E- 800
`:i...J
`..- E
`600
`Al-
`Q) 0 ~g 400
`t..-
`200
`ns-
`CL.
`0
`
`CP3
`
`A
`
`n=2 B
`
`n=3
`
`C
`
`GV CP1 CP2 CP3 CP4
`
`GV CP1 CP2 CP3 CP4
`
`CP1 CP2 CP3 CP4
`
`CP4
`
`l
`
`l
`
`100µm
`
`100µm
`
`FIGURE 2. Particles per milliliter (2-1
`µm) of bevacizumab directly from the
`original glass vial (GV) and repackaged
`bevacizumab ordered from four exter(cid:173)
`nal CPs. (A) First batch; (B) second
`batch tested on receipt (n = 3); (C)
`second batch tested within 14 days of
`the 3-month expiration date (n = 2).
`Representative images of CP3 and CP4
`samples in the first batch (bottom:
`1:30 dilution; arrows: particles).
`
`The same samples of repackaged bevacizumab were an(cid:173)
`alyzed by SE-HPLC for monomer levels and soluble aggre(cid:173)
`gates (Figs. 4, 5). Representative chromatograms are shown
`
`-C:
`.... .c
`C: <I)
`:::, N o·-o (/)
`~:3
`... _
`~E
`n, 0 a. 0
`"""" -
`0
`
`140
`120
`100
`80
`60
`40
`20
`0
`
`C: <I)
`
`-C:
`.... :.c
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`"""" -
`
`0
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`CP3
`
`-
`-
`-
`
`Sample1
`Sample2
`Sample3
`
`5
`
`10
`Diameter(µm)
`
`15
`
`20
`
`CP1
`
`-
`-
`-
`
`Sample1
`Sample2
`Sample3
`
`5
`
`10
`
`15
`
`20
`
`Diameter(µm)
`
`FIGURE 3. Particle size distribution (2-1 µm) of repackaged bevaci(cid:173)
`zumab from three syringes from CP3 (first batch) and CPl (second
`batch, tested on receipt).
`
`in Figure 4, with the peaks for high-molecular-weight
`(HMW) species 1 and 2 labeled (HMWI and HMW2) in the
`Figure 4 inset. The weight-averaged molecular mass of the
`species in the chromatogram peaks was estimated with an
`online light-scattering detector. Based on analysis of the
`SE-HPLC results for all samples studied by this method (data
`not shown), we determined that the HMWI species were
`dimers to tetramers and the HMW2 species were composed
`of higher-order oligomers ranging from octamers to decam(cid:173)
`ers.
`SE-HPLC analysis showed that there was substantial monomer
`loss in the repackaged bevacizumab in batch one from CP2 and-3,
`whereas the levels in samples from CPI and -4 were similar to that
`for bevacizumab taken directly from the drug product vials (Figs.
`5A-C). For the repackaged bevacizumab in batch 2, the monomer
`level in samples from all the external compounding pharmacies
`was about the same as that measured in the bevacizumab taken
`directly from the drug product vials. In the samples in batches one
`and two, the levels of HMWI ranged from approximately 2% to
`4% and the levels of HMW2 from approximately 0.2% to 0.4%,
`respectively (Fig. 5).
`Compared with the samples of repackaged bevacizumab
`obtained in the first batch, the range of particle counts was
`narrower among all the samples in the second batch when they
`were tested immediately on arrival (Fig. 2B). However, particle
`counts varied substantially between the samples taken from
`individual syringes in the second batch of repackaged bevaci(cid:173)
`zumab from CPI (Fig. 3). When we tested the second-batch
`samples that were stored at 4°C until 14 days before the
`3-month expiration date, there was an increase in the total
`particles per milliliter in samples from CP2 and CP4 (Fig. 2C).
`There were no substantial changes in the levels of monomer or
`high-molecular-weight bevacizumab in the stored samples
`(Figs. 5D-F).
`
`Controlled Laboratory Studies of
`Repackaged Bevacizumab
`Three-Month Incubation Study. Laboratory samples of
`repackaged bevacizumab and placebo had initial (after 2 hours
`at 4°C, used as time-0 data) particle counts, respectively, of
`283,675 ±: 60,494/mL and 492,314 ±: 389,361/mL (Table 2). In
`
`Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932965/ on 10/09/2017
`
`Regeneron Exhibit 1025.005
`
`

`

`1028 Liu et al.
`
`IOVS, February 2011, Vol. 52, No. 2
`
`400
`
`380
`
`JI ~360
`
`N
`~ 340
`
`320
`
`300
`
`16.5
`
`17.0
`
`Eluti on time (Minut e)
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`0
`co
`N
`.c
`<(
`
`Glass vial
`CP2
`CP3