ARTICLE IN PRESS

Journal of Pharmaceutical Sciences 000 (2023) 1−9

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences

jo urn a l h om ep ag e : w ww.jp harms ci.o rg

Pharmaceutical Biotechnology

Effect of Various Silicone Oil And Tungsten Levels on the Stability

of a Monoclonal Antibody in Nine Commercially Available
Prefilled Syringes
Markela Ibo Murphy*, Jesse A. Leissa, Sandra B. Plata, Amy L. Chamberlain,
Sajal M. Patel
Dosage Form Design & Development, BioPharmaceuticals R&D, AstraZeneca, Gaithersburg, MD, USA

A R T I C L E I N F O A B S T R A C T

Article history: Prefilled syringes are widely used as a primary container for therapeutic proteins because they are more con-
Received 1 July 2022 venient than glass vials. The stability of biologic molecules can be affected by different syringe materials and
Revised 13 March 2023 techniques, such as silicone oil levels and coating method, amount of tungsten remaining in the glass barrel
Accepted 13 March 2023
after using a tungsten pin to create the needle hole, and end of the syringe, which can be Luer locked or pre-
Available online xxx
staked with a needle. We investigated the impact of these parameters by using a monoclonal antibody to col-
lect the antibody’s stability profile and the prefilled syringes’ functionality data. Silicone oil levels had no
Keywords:
impact on aggregation levels, and particle counts were lowest for silicone oil−free syringes. Functionality
Protein stability
Prefilled syringe
performance was similar and did not change throughout all stability time points for all syringe configura-
Silicone oil tions. The break-loose force for Ompi syringes was initially lower and increased over time to align with those
Tungsten of the other configurations, all of which remained well below 25 N. Tungsten contaminants and agitation
Glide force stress from shipping studies did not impact quality attributes. This work can help guide the development of
Particle size similar products in prefilled syringes to ensure selection of the primary container that provides adequate sta-
Protein formulation bility for the protein, as well as maintain the desired functionality features over the shelf life of the drug
product.
© 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Introduction product viscosity.5 In contrast, the holdup volume in a prefilled

As of 2022, more than 1500 marketed biopharmaceutical paren-
teral medications in the United States are approved for administra-
tion via injection for various indications, including cancer, metabolic
disease, infections, and immune and inflammatory conditions.1 Most
of these products are stored in vials, followed by prefilled syringes,
cartridges, and ampules. The use of prefilled syringes has grown sub-
stantially in recent years, especially for those containing medications
for chronic diseases, because they enable self- or at-home adminis-
tration and therefore offer increased convenience for both patients
and health care providers.1,2 Prefilled syringes as a primary container
also offers additional advantages for the patient as well as the manu-
facturer, including increased product competitiveness, ease of admin-
istration, reduced chances of microbial contamination during dose
preparation, and reduction in dosing errors3,4. The U.S. Pharmacopeia
(USP) requires 20 or 24% overfill for a 0.5-mL fill in a vial based on
* Corresponding author at: Markela Murphy, AstraZeneca, One MedImmune Way,
Gaithersburg, MD 20878.
E-mail address: Markela.Murphy@AstraZeneca.com (M.I. Murphy).
syringe is generally an order of magnitude lower than that in a glass
vial.
Although prefilled syringes as a primary container and drug deliv-
ery system have advantages over vials and disposable syringe sys-
tems, their use also adds complexity for the drug product
development team, which must ensure that the sensitive biologic
molecule is compatible with all the syringe components and materi-
als that come in contact with the product through its entire shelf life.
Syringes are traditionally composed of a Type I glass barrel, an elasto-
mer stopper, a plunger rod, a needle shield, and a staked needle that
is secured with an adhesive to the glass barrel. Syringes without a
staked needle have a Luer lock tip to which the needle can be
attached, which is needed when the product is to be delivered intra-
muscularly and various needle lengths are required based on the
patient’s weight.6
The inner surfaces of the glass barrel and the elastomer stopper
are coated with a layer of silicone oil that serves as a lubricant, allow-
ing the smooth motion of the stopper across the length of the syringe
barrel and lowering the injection force during drug administration.
Improving syringe functionality through siliconization, however,

https://doi.org/10.1016/j.xphs.2023.03.009
0022-3549/© 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.
 ARTICLE IN PRESS
2 M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9

may cause stability issues for the biologic molecule. Studies have commercially available prefilled syringes. In particular, the objective
shown that silicone oil droplets may migrate from the syringe barrel of this work was to understand the effect of agitation and various
into the protein formulation, increasing the number of subvisible par- syringes manufactured with different specifications, such as amount
ticles in the drug product during storage and transportation7. In addi- of silicone oil, tungsten levels, and syringe configuration, on protein
tion, the protein molecules can migrate at the water-oil or air-water stability at various time points by performing visual inspection, mea-
interfaces, and the latter may cause conformational perturbation that suring particle concentrations with microflow imaging (MFI), and
leads to unintended protein aggregation.8 Subvisible particle concen- evaluating aggregation levels with size exclusion chromatography. In
tration is a major concern during drug product development; the addition, break-loose force (the maximum force required to break
United States Pharmacopeia (USP) Standards 788 and 787, the Euro- the static friction of the stopper) and glide force (the force required
pean Pharmacopoeia (EP) 2.9.19, and the Japanese Pharmacopoeia to maintain stopper movement once static friction has been over-
(JP) 6.07 specify that the acceptable particle concentration for particle come) were measured to evaluate the impact of the different siliconi-
size categories of ≥10 mm and ≥25 mm should not exceed 6000 and zation methods and the amount of silicone oil on syringe
600, respectively, for parenteral drug products.9-12 Syringe manufac- functionality.
turers have addressed these concerns by establishing specifications
for the amount of silicone oil present in prefilled syringes and by
optimizing various siliconization methods.
Experimental Section
Two siliconization methods are commercially available: sprayed-
on and sprayed-on followed by a baked-on step. In the sprayed-on
Stability Studies
coating method, silicone oil is applied by a fixed or diving nozzle; the
latter generates a more uniform coating across the entire length of
A monoclonal antibody (mAbA) was formulated at a concentra-
the syringe barrel. Baked-on coating is applied by a sprayed-on pro-
tion of 100 mg/mL with surfactant at industry-relevant concentra-
cess followed by a baked-on layer of silicone oil. This process pro-
tion. The protein solution was sterile filtered through a 0.2-mm
vides a layer that is better adhered to the glass barrel than the un-
Millipore filter and hand filled to either 1.0 or 0.5 mL into each of
baked silicone oil layer, thereby reducing the amount of silicone oil
nine different ready-to-use for further manufacturing syringe config-
that can migrate into the protein formulation over time.7,13 Protein
urations (Table 1). A 1-mL fill in a 2R vial configuration was used as
instability in the presence of silicone oil can be also addressed by
control. BD Neopak (Becton Dickinson, Franklin Lakes, NJ, USA), BD
using formulation excipients such as surfactants, which migrate at
Hypak (Becton Dickinson), Gx RTF (Gerresheimer, Bu € nde, Germany),
the interfaces and inhibit the migration of protein molecules.14,15 In
and Nuova Ompi (Nuova Ompi, Padova, Italy) glass syringes were
addition, some manufacturers have introduced silicone oil−free
stoppered by using a vacuum stoppering instrument (Rormac Pack-
syringe barrels by using a novel fluoropolymer surface on the stop-
aging Systems, Reading, PA, USA), and SyriQ (Schott, Mainz, Ger-
per, which allows for the injection force to remain within specifica-
many) glass syringes with Improject stoppers (WL Gore, Elkton, MD,
tions for a manual injection.16
USA) were individually stoppered by hand. This syringe configuration
Agitation during the transportation of the drug product is another
does not allow for vacuum stoppering due to the lack of silicone oil in
factor that needs to be taken under consideration. Movement of the
the syringe barrel and stopper. All syringe stoppers were received
air bubble between the solution and the stopper during agitation
sterilized and ready to use, and no further treatment was imple-
increases the amount of subvisible particles as compared to no air
mented. Type 1 glass tubing vials (2R) and 13-mm serum stoppers
bubble syringes, and it depends on the syringe coating method. 17
were obtained from West Pharmaceutical Services (Exton, PA, USA).
Shipping stress may also have an impact on product quality by induc-
All primary container systems were subjected to 2 £ shipping simu-
ing the aggregation of proteins of various sizes, from subvisible to vis-
lations, which corresponds to 1200 miles through truck and air trans-
ible particles. A shipping simulator vibration table has been used to
portation, before being stored at 2−8°C for up to 12 months 25°C for
mimic the effect of shipment on the formation of subvisible particles
up to 6 months, or 40°C for up to 1 month. At each time point, five of
in various configurations of vials and prefilled syringes.18
the 1-mL filled syringes and seven of the 0.5-mL filled syringes were
Tungsten species is another material that comes in direct contact
pooled for analysis. The air bubble for all syringe configurations was
with the drug product throughout its shelf life when a prefilled
less than 4 mm in diameter except for the Gore syringes, which had
syringe is used as the primary container. The hole in the syringe is
an air bubble of approximately 5 mm in diameter. Air bubbles in all
created by using a tungsten pin against the glass tubing that is used
syringes were able to move along the syringe barrel during agitation
to form the glass barrel. During this process, very high temperatures
or syringe movement. Levels of silicone oil and tungsten for each
and mechanical stresses are applied, which results in sublimation
syringe configuration were set according to the vendors’ specifica-
and deposition of tungsten material on the inner surface of the bar-
tions (Table 1).
rel.19 After the hole is formed, the syringe undergoes a rinsing step,
after which traces of the tungsten remain. Tungsten polyanions have
been demonstrated to electrostatically interact with protein, leading
to particle and protein precipitation.20 Particle formation in the pres- Visual Inspection
ence of tungsten polyanions has been shown to depend on formula-
tion pH, tungsten concentration, protein properties, manufacturing After the syringes were equilibrated to room temperature, all
processes, and protein-to-tungsten ratio.21-23 Manufacturers have syringes were visually inspected under a light box with a white and a
addressed potential tungsten-induced protein degradation by setting black background for the presence of visible particles, color, and clar-
specifications for the highest amount of tungsten species that can be ity. The lux of the lamp used in the light box ranged between 3000
present in a syringe barrel. Another approach is to use metal-free and 3400 lx. The assessment was performed by comparing the sam-
pins to generate the needle hole. Under any circumstances, the devel- ples in the syringes to a range of reference standards. The particle
opment team must perform the necessary studies to ensure that the standards were prepared in house by using barium sulfate stock solu-
particular formulation, protein molecule, and prefilled syringe config- tion (Sigma-Aldrich, Darmstadt, Germany). The color standards
uration are compatible. (Sigma-Aldrich) were purchased ready to use, and clarity standards
In this work, we evaluated the stability of a monoclonal antibody (Hach-Lange, Loveland, CO, USA) were prepared according to the
(mAb) during storage under different conditions in various manufacturer’s recommendations.
 ARTICLE IN PRESS
M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9 3

MFI
Nuova Ompi 1 mL Long

Ompi EZ-Fill/Luer lock

Datwyler, Omniflex
Subvisible particle concentration was measured with an MFI 5200

Diving nozzle,
West 7025/65

sprayed-on
series instrument (Protein Simple, San Jose, CA, USA) equipped with a

silicone oil
0.7 mg max
100-mm flow cell and controlled by the MVSS Software (version

Unknown

≤4 mm
5.1.076, S/N 1999) supplied with the instrument. The flow cell was
cleaned between samples with 1% (v/v) Tergazyme solution (Alconox,
New York, NY, USA) and rinsed with purified water. An “optimize
illumination” step was run prior to each measurement to correct the
system threshold. Samples were measured by first flushing the flow
cell with 0.2 mL of sample to equilibrate the system, followed by
0.3 mL of sample for counting, and particle sizes were calculated as
Schott/ Luer lock

equivalent circular diameter. Because no aspect ratio filter was

West 7025/65

applied, measurements included protein particles as well as silicone

Unknown

oil droplets.

HPSEC

Monomer, aggregated, and fragmented proteins were quantified

with an Agilent (Santa Clara, CA, USA) high-performance size exclu-
sion chromatography (HPSEC) system with ultraviolet absorbance at
Gerresheimer RTF/Luer lock Schott SyriQ/ staked needle

a wavelength of 280 nm. The mobile phase consisted of 0.1 M sodium

phosphate, 0.1 M sodium sulfate, and 0.05% (w/v) sodium azide at pH
6.8 in highly purified water. The protein was separated over a
Gore 1 mL Long

Silicone oil free

Gore Improject

7.8 £ 300 mm2 TSKgel G3000SWXL column (Tosoh Bioscience, Tokyo,

Japan) with a 6.0 £ 40 mm2 TSKgel SWXL guard column at a flow rate
Metal free

of 1.0 mL/min. Protein samples were diluted from 100 mg/mL to

10 mg/mL with phosphate buffer prior to analysis, and 25 mL of sam-
NA

NA

»5−6 mm

ple was injected into the HPSEC system. The percentages of each spe-
Metal free 0.05−0.5 ppm

cies, monomer, aggregates, and fragments were calculated as the

4023/50 FluroTec coated
Gerresheimer 1 mL Long

Diving nozzle, baked-on

area under the curve of each species divided by the total area.
West 7025/65

Some specifications were unavailable because the vendor declined to share information for proprietary reasons.

Break-Loose and Glide Force Measurements

silicone oil
Unknown

Break-loose and glide forces were quantified to assess the influ-

ence of syringe and stopper surface properties and lubrication on the
sprayed-on

injection performance of each configuration. Both break-loose and

Fixed nozzle,

silicone oil
NMT 1 ppm Potential for up to 10 ppm
0.04 mg max 0.12 mg max 0.7 mg max

glide forces were measured with a 5542 series electromechanical sin-

Abbreviations: max, maximum; NA, not applicable; NMT, not more than; PFS, prefilled syringe.

gle-column compression/tension testing instrument (Instron, Nor-

wood, MA, USA) on all syringe configurations at each time point
Characteristics of various commercially available syringe systems evaluated in this worka.

West 7025/65

(n = 3). Each syringe was equilibrated to room temperature and then

Diving nozzle, baked-on

placed vertically in the instrument with the needle (25G £ 5/8") fac-
BD Hypak 1 mL Long

ing downward. The break-loose and average glide force was deter-
mined with a 100 N load cell system at a controlled rate of
silicone oil
BD/Luer lock

260 mm/min until a maximum force of 40 N was achieved, indicating

that the total amount of liquid had been expelled from the syringe
FM30/0

barrel.
Diving nozzle, sprayed-on

Shipping Simulator Vibration Table

BD Neopak 1 mL Long

Barrel manufacturer/ tip type BD/staked needle

BD 4023/50 Fluro

The shipping simulator (Model 1000 Vibration Test System; Lans-

silicone oil
Tec coated

mont, Monterey, CA) was used to mimic the real-time shipment

Silicone oil specification limits 0.4 mg max

stresses that are exerted on the protein and to simulate the bubble
Tungsten specification limits 0.5 ppm

movement in the prefilled syringes during distribution of drug prod-

NA

uct. A 12-step profile with air and truck level II intensity was used to
≤4 mm

simulate the vibration profiles that would be encountered during

two round trips from Maryland to California, or 96 hours of real-time
Silicone oil application

shipment. This duration was compressed to 24 hours of simulation

PFS characteristics

time, during which truck-air-truck level II was repeated four times

(two round-trip shipments).18 Samples were stored in syringe tubs,
Rubber tip cap

Bubble size

which were then placed inside an insulated transportation tote with

Stopper

cold packs to maintain a temperature of 2−8°C. The transportation

Table 1

tote was secured to the vibration table of the shipping simulator

a

before a run was initiated.

 ARTICLE IN PRESS
4 M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9
Results
Inspection of Visible Particles
Upon visual inspection, all the syringe systems appeared to be
practically free from visible particles, slightly opalescent, and slightly
yellow from time 0 up to 12 months of storage at 2−8°C. The visual
inspection was conducted according to USP chapter 790.24
Physical Protein Stability by HPSEC
Monomer purity and aggregate and fragment levels were deter-
mined by HPSEC for mAbA in all nine syringe configurations and the
2R vial control sample. The representative stability data are pre-
sented in Fig. 1, which shows HPSEC profiles obtained at various sta-
bility time points during storage at 2−8°C. Time 0 on the graphs
represents the data obtained before and after 2 £ simulated ship-
ments. Monomer purity loss and aggregation and fragmentation rates
for mAbA were similar among the nine syringe configurations and
were not affected by the various levels of silicone oil, tungsten, and
agitation induced during 2 £ shipping simulations. The stability pro-
file of mAbA stored in the 2R vial was similar to that of the molecule
stored in the nine syringe systems. An accelerated stability study was
conducted, and Table 2 shows the HPSEC results for mAbA after
2 £ shipping simulations and storage at 40°C for 1 month. The frag-
mentation rate of the sample in the 2R vial was 2.3%, whereas the
highest fragmentation level observed for the Gerresheimer syringes
was 3%. HPSEC results from the accelerated stability study indicated
that fragmentation was the primary degradation pathway for all nine
syringe configurations and was similar to the control sample in the
2R vial. The two different fill volumes also had no impact on the
physical stability of mAbA during long-term storage at 2−8°C or dur-
ing the accelerated stability study, as determined by HPSEC.
Fig. 1. Monomer, aggregate, and fragmentation content obtained by HPSEC for mAbA in 10 different primary container configurations during storage at 2−8°C for up to 12 months.
(a−c) 1 mL fill volume; (d−f) 0.5 mL fill volume.
Impact of Shipping Agitation on Subvisible Particle Levels Measured by
MFI
Subvisible particle counts in samples upon filling, indicated as time
0, and after 2 £ shipping simulations were measured by MFI (Fig. 2).
Particles in the ≥2 mm bin were studied to gather information about a
potentially important size range, although this is not required by the
U.S. Pharmacopeia. Fig. 2 shows that agitation did not affect the parti-
cle counts in any of the syringe configurations with or without silicone
oil coating, which are the syringes with the Gore stoppers.
Impact of Siliconization on Subvisible Particle Levels Measured by MFI
Subvisible particle counts in samples from various stability time
points were measured by MFI for all 10 primary container configura-
tions (the nine syringe configurations plus the control 2R vial) with
diverse silicone oil specification limits. The silicone oil levels present in
the syringe glass barrel were not measured, therefore levels cited in
this manuscript are the specification limits provided by the vendors.
Data for samples stored at 2−8°C are shown in Fig. 2, and data for sam-
ples stored at 25°C are shown in Fig. 3. We evaluated the impact of sili-
cone oil level on particle counts for the three bin sizes (≥2 mm,
≥10 mm, and ≥25 mm). The MFI data showed that the syringes with
no silicone oil on the glass barrel (the Schott syringes with the Gore
stopper) had lower counts than the syringe configurations with some
level of silicone oil. The Gerresheimer syringes with baked-on silicone
oil, which have an unknown level of silicone oil, had particle counts
similar to those of the syringes with sprayed-on silicone oil.
Figs. 2 and 3 show data for the two syringe systems, Ompi and BD
Hypak, with the highest silicone oil specification limits of 0.7 mg. The
Ompi system produced the highest particle counts for both fill vol-
umes of 1 mL and 0.5 mL stored at 2−8°C and 25°C. Both of the
syringe systems with the 0.7 mg silicone oil specification limits were
siliconized by the sprayed-on method, so we speculate that the Ompi
 ARTICLE IN PRESS
M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9 5

Table 2
HPSEC data of mAbA obtained with 10 different primary container configurations over storage of up to 1 month at 40°C stability condition.

Configuration Fill volume (mL) HPSEC data

2 £ Shipping 1 month

Container/tip type SO amount (mg) % Aggregates % Monomer % Fragments % Aggregates % Monomer % Fragments

2R vial NA 1 0.8 98.3 0.9 1.1 95.6 3.2

Gore/SN 0 0.8 99.0 0.2 1.1 96.2 2.7
Gore/LL 0 0.8 99.0 0.2 1.0 96.4 2.5
BD Hypak/LL 0.04 0.8 98.4 0.8 NT NT NT
BD Hypak/LL 0.12 0.8 98.2 0.9 1.1 95.9 3.0
BD Neopak/SN 0.4 0.8 99.0 0.2 1.0 96.5 2.5
BD Hypak/LL 0.7 0.8 98.3 0.9 1.1 96.2 2.7
Ompi/LL 0.7 0.9 98.2 0.9 1.1 95.9 3.0
Gerresheimer tungsten free/LL Unknown 0.8 99.0 0.1 1.1 95.8 3.1
Gerresheimer w/ tungsten/LL Unknown 0.8 99.0 0.1 1.1 95.9 3.1
Gore/SN 0 0.5 0.8 99.0 0.2 1.1 96.2 2.7
Gore/LL 0 0.8 99.0 0.2 1.3 96.2 2.5
BD Hypak/LL 0.04 0.8 98.4 0.8 NT NT NT
BD Hypak/LL 0.12 0.9 98.2 0.9 1.1 95.9 3.0
BD Neopak/SN 0.4 0.8 99.0 0.2 1.0 96.5 2.5
BD Hypak/LL 0.7 0.8 98.3 0.9 1.1 96.2 2.7
Ompi/LL 0.7 0.8 98.2 0.9 1.1 96.0 2.9
Gerresheimer w/ tungsten/LL Unknown 0.8 99.0 0.1 1.1 95.8 3.1
Abbreviations: LL, Luer lock; NT, not tested; SN, staked needle; SO, silicone oil.

syringes contained higher levels of silicone oil than the BD Hypak at
the time that they were filled with the mAbA formulation. The manu-
facturer’s specification for the maximum amount of silicone oil that
can be present in the glass barrel is 0.7 mg; however, during storage
of unused, empty syringes, the silicone oil migrates outside the
syringe barrel, and therefore the silicone oil level is reduced through-
out the storage period until they are filled.
Syringes with the three lowest silicone oil specification limits had
similar particle counts over the storage period at 2−8°C and 25°C.
Particle counts were higher in these syringes than in the non-silicon-
ized syringes and were lower than in the Ompi configuration. Sur-
prisingly, the BD Hypak, which had a silicone oil level of 0.12 mg, had
higher particle counts than the rest of the configurations, which had
similar silicone specification limits but only for the 0.5 mL fill. We
speculate that during the stoppering process for this particular
syringe configuration, the amount of silicone oil was scraped off from
the glass barrel and migrated into the formulation solution, and this
amount was greater in the 0.5 mL fill than in the 1 ml fill because the

Fig. 2. Subvisible particle counts of ≥2 mm, ≥10 mm, and ≥25 mm measured by MFI for mAbA stored in 10 different primary container systems at 2−8°C for up to 12 months. (a−c)
1 mL fill volume; (d−f) 0.5 mL fill volume.
 ARTICLE IN PRESS
6 M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9

Fig. 3. Subvisible particle counts of ≥2 mm, ≥10 mm, and ≥25 mm measured by MFI for mAbA stored in 10 different primary container systems at 25°C for up to 6 months. (a−c)
1 mL fill volume; (d−f) 0.5 mL fill volume.

stopper traveled a longer distance along the syringe barrel for the
0.5 mL fill than for the 1.0 mL fill.
Syringe Functionality and Performance
Break-loose and glide force measurements were collected for the
nine different syringe configurations at time 0, after the 2 £ shipping
simulations, and during long-term storage at 2−8°C for up to 12
months (Fig. 4) and at 25°C for up to 6 months (Fig. 5). The break-
loose values for eight of the nine syringe configurations remained
consistent during storage. The break-loose values of the Ompi config-
uration at the beginning of the study were lower than the rest of the
configurations tested, increasing from 2 N to 7 N during storage for
12 months at 2−8°C and from 2 N to 11.6 N at 25°C for fill volumes of

Fig. 4. Comparison of break-loose and average glide force among the nine different syringe configurations with various silicone oil levels and two different syringe tips (staked nee-
dle and Luer lock). The sample syringes were stored at 2−8°C for up to 12 months. (a−b) 1 mL fill volume; (c−d) 0.5 mL fill volume.
 ARTICLE IN PRESS
M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9
Fig. 5. Comparison of break-loose and average glide force among the nine different syringe configurations with various silicone oil levels and two different syringe tips (staked nee-
dle and Luer lock). The sample syringes were stored at 25°C for up to 6 months. (a−b) 1 mL fill volume; (c−d) 0.5 mL fill volume.
1 mL and 0.5 mL, respectively. The reason for the increased break-
loose force of the Ompi syringes may be that the compressive force
exerted in the region between the stopper and the glass barrel during
storage causes the silicone oil in that region to be pressed out.7
The Gore syringe configurations had the highest break-loose val-
ues, ranging from 7 N to 10 N, than the other configurations, whose
values ranged from 3 N to 7 N; however, the break-loose values for
all the configurations were below the target values, 25 N, for func-
tional performance. The glide force values of all the syringe configu-
rations remained consistent during storage at 2−8°C and 25°C; the
Ompi syringes had the lowest values at 3 N, and the Gore configura-
tions had the highest values at 7.5 N. The functionality trends for the
1 mL and 0.5 mL fills for all nine syringe systems were similar.
Discussion
We conducted a comparative study to reveal any differences in
monomer purity loss and aggregation level, subvisible particle gener-
ation, and functionality measurements among 10 different primary
container systems (nine syringe configurations and a 2R vial as con-
trol), using a mAbA formulation. The nine syringe systems had six dif-
ferent silicone oil levels based on specification limits, ranging from
0 mg to a maximum of 0.7 mg. The silicone oil application methods
used included a fixed or diving nozzle with baked-on or sprayed-on
silicone oil. The syringe configurations had a variety of tungsten lev-
els, from tungsten-free syringes, for which a ceramic instead of a
tungsten pin was used to create the needle hole on the syringe barrel,
to a potential maximum of 10 ppm. For some of the syringes, the con-
tent of tungsten was not known (Table 1). Eight of the syringe config-
urations had two fill volumes, 1 mL and 0.5 mL, whereas the
Gerresheimer tungsten-free and the 2R vial had only a 1 mL fill vol-
ume. The syringe systems also had either a Luer lock or a staked nee-
dle tip. All the drug product presentations discussed here had similar
7
visual appearances, being practically free from visible particles,
slightly yellow, and slightly opalescent.
The stability of the mAbA was characterized with respect to
monomer purity loss and aggregation level in a control 2R vial and in
prefilled syringes that were siliconized at maximum specification
limits of 0, 0.04, 0.12, 0.4, and 0.7 mg of silicone oil per syringe. Sili-
cone oil may be released from the syringe barrel into the formulation
solution or may adhere to the glass surface, depending on the silico-
nization method (sprayed-on or baked-on); in both cases, there is a
silicone oil−water interface. As a consequence, protein may adsorb to
the silicone oil−water interface, leading to protein aggregation.8,25
The silicone oil levels studied here had no impact on aggregation lev-
els or purity loss after storage at 2−8°C for 12 months or at 40°C for 1
month. Similar results have been reported by Bai et al., who found
that silicone oil levels did not affect the rate of aggregate formation.26
Basu et al. reported that agitation in the presence of siliconized beads
can sometimes increase aggregation of an IgG1 molecule in the
absence of surfactant 27.To ensure protein stability in the primary
containers during transportation, the drug products were exposed to
agitation stresses during 2 £ shipping simulations. After agitation, sil-
icone oil level had no impact on monomer purity or protein aggrega-
tion, as the level of aggregation for the 2R vial and the Gore syringes
that contained no silicone oil were similar to that of the syringes con-
taining the highest amount (0.7 mg) of silicone oil based on the speci-
fication limits Similar results were reported in a previous study in
which agitation in the presence of silicone oil had no obvious effect
on protein aggregation.26 It may be that the presence of excipients
such as polysorbate 80 prevents the exposure of protein to the sili-
cone oil−water interface, which in turn prevents the silicone oil level
from having an impact on protein aggregation, even upon
agitation.14,15 Protein aggregation and loss of monomer purity has
been associated with tungsten in prefilled syringes manufactured
with a tungsten pin.20,22,23,28 Jiang at el. found that aggregation of a
mAb by tungsten species was observed only at tungsten levels of
 ARTICLE IN PRESS
8 M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9
greater than 10 ppm and less than pH 6.20 In line with previously
published results,21-23 the aggregation level, in this study, in the
syringe with the highest potential amount of tungsten was compara-
ble to that in the metal-free syringe and the 2R vial. Therefore, the
tungsten levels studied here had no effect on mAbA aggregation or
monomer purity loss.
Subvisible particle levels in parenteral drug products need to be
monitored and quantified as a regulatory requirement because they
may cause an unintended immune response. In this work, we evalu-
ated the impact of silicone oil levels based on specification limits, agi-
tation stress, silicone coating method, and fill volume on the
generation of subvisible particles in several configurations of prefilled
syringes. We found that the Ompi syringes, which had the highest sil-
icone oil specification limits, had the highest particle counts. This
finding is in agreement with results reported by Bai at el., who
detected higher particle counts in syringes with the highest amount
of silicone oil.26 In our study, subvisible particle counts of samples in
all 10 primary containers were similar before and after agitation,
indicating that the agitation that occurred during the 2 £ shipping
simulations did not disturb the silicone oil that was attached to the
syringe glass barrel. This is in agreement with the results of a study
in which agitation induced particle formation depended on the integ-
rity of the lubricant coating, where enhanced integrity resulted in
fewer particle counts.7
The impact of the siliconization method has been extensively
reviewed in other studies, which have reported that subvisible parti-
cle counts for syringes with sprayed-on silicone were higher than
those for syringes with baked-on silicone.7,17,29 In contrast, our
results showed that the silicone oil coating method did not have a
significant impact on subvisible particle counts, as similar levels were
detected in syringes coated by the baked-on and the sprayed-on
method. Fill volume had an impact on particle formation only for the
syringe configuration that contained 0.12 mg of silicone oil, whereas
the drug product with the 0.5 mL fill had higher particle counts than
the drug product with the 1 mL fill.
Siliconization of the syringe glass barrel is primarily performed to
enable the stopper to glide smoothly along the entire length of the
syringe and to minimize break-loose and gliding forces over the shelf
life of the product. The aim of siliconization is to achieve a uniform
coating by using a minimum quantity of silicone oil. In this work, we
explored various silicone oil levels based on specification limits and
coating technologies involving sprayed-on or baked-on silicone oil.
The syringes without a siliconized glass barrel, such as the Gore
syringe, had higher break-loose force than the syringes with a silicon-
ized glass barrel, which was expected and has been observed in other
studies.17 The break-loose forces of the syringes with silicone oil
specification limits ranging from 0.04 mg to 0.7 mg all had similar
values except for the Ompi configuration, which had a lower break-
loose force at time 0. At 12 months, the break-loose force of the Ompi
configuration had reached a level similar to those of the rest of the
siliconized syringes. This finding can be attributed to the compressive
force exerted in the region between the stopper and the glass barrel
which causes the silicone oil in that region to be is pressed out and
allows the rubber stopper to come into contact with the naked glass
barrel over the storage period.7 This phenomenon has been observed
to a much lesser extent for baked-on siliconized syringes, for which
the break-loose force remained practically constant over the entire
storage period.13 In our study, the glide forces were similar for all the
silicone oil specification limits ranging from 0 to 0.7 mg except for
the Ompi system, which had the lowest glide force throughout the
duration of the stability period of 12 months. In contrast, other stud-
ies have shown that the baked-on siliconized syringes had higher
glide forces than sprayed-on siliconized syringes. This difference is
due to the high temperatures used during the baking process, which
may alter the lubrication properties of silicone oil.30
Conclusions
The aim of this work was to understand the effects of various
parameters of prefilled syringe systems on the stability of protein
therapeutics and their contribution to the functionality of the pre-
filled syringes. To this end, we conducted a series of comparative
studies on the silicone oil level, siliconization technology, residual
tungsten amount, tip of the syringe (Luer lock or staked needle), fill
volume, agitation stresses, and temperature stress conditions to
assess any incompatibilities of the mAbA with the syringe parameters
that are necessary in prefilled syringe systems.
Our results showed that silicone oil level had no impact on protein
aggregation, even under agitation and temperature stress conditions.
Similarly, protein stability in syringes with the highest level of tung-
sten specifications, up to 10 ppm, was comparable with that in tung-
sten-free syringes. Theoretically, the residual amount of tungsten
would be higher in a Luer lock syringe than in a staked needle
syringe, because in the latter configuration, part of the syringe hole is
covered by the glue that is used to stabilize the needle, so some
amount of tungsten is buried underneath the glue layer. In this study,
however, syringes with the two different tips had similar stability
profiles. In addition, the holdup volume in the Luer lock syringes was
approximately seven times higher than that in the staked needle
syringes (data not shown). Subvisible particle generation in all 10 pri-
mary containers was monitored and quantified, and the results
showed that The syringes with sprayed-on silicone oil had subvisible
particle trends that were similar to those of the syringes with baked-
on silicone oil, except for the Ompi syringes, which had the highest
levels of subvisible particle counts for most of the stability time
points. Fill volume made a difference only for the BD Hypak, which
had a maximum silicone oil level of 0.12 mg, whereas syringes with a
0.5 mL fill volume had higher particle counts than those with a 1 mL
fill volume. Functional performance and break-loose and glide forces
were measured at predetermined stability time points, and the data
showed that the silicone oil−free syringes, which had the Gore stop-
pers, had the highest break-loose force values, but their glide force
values were similar to those of the rest of the configurations. The
syringe system with the Gore stoppers were manually stoppered
instead of vacuum stoppered like the rest of the syringes, which could
impact technology transfer activities; this can be considered a draw-
back for this particular syringe configuration. In addition, the Ompi
syringe system showed a significant increase in the break-loose force
with aging and temperature, which was attributed to the sprayed-on
coating, although it showed the lowest glide force among all the con-
figurations.
Overall, similar performance was shown by all the syringe config-
urations except the Ompi syringes, which showed a change in func-
tionality with time and temperature. In addition, the syringes with
the Gore stoppers could not be vacuum stopped according to the
manufacturer’s recommendations, and this needs to be taken under
consideration when choosing the right primary container for the
drug product.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Supplementary Materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.xphs.2023.03.009.
 ARTICLE IN PRESS
M.I. Murphy et al. / Journal of Pharmaceutical Sciences 00 (2023) 1−9
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