Comparison of flow and batch polymerization processes for production of vinyl ether terpolymers for use in the delivery of siRNA

Article abstract of DOI:10.1002/pola.27094

Synthetic polymers represent a modifiable class of materials that can serve as adjuvants to address challenges in numerous biomedical and medicinal chemistry applications including the delivery of siRNA. Polymer-based therapeutics offer unique challenges in both synthesis and characterization as compared to small molecule therapeutics. The ability to control the structure of the polymer is critical in creating a therapeutic. Reported herein, are batch and flow polymerization processes to produce amphiphilic terpolymers through a Lewis acid BF₃OEt₂-catalyzed polymerization. These processes focus on controlling reaction variables, which affect polymer structure in this rapid, exothermic, nonliving cationic polymerization. In addition to analytical characterization of the polymers, the in vivo activity of the polymer-siRNA conjugates is also highlighted - demonstrating that the method of synthesis does affect the in vivo activity of the resulting polymer conjugate. 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 1119-1129 The ability to control the character of the polymer structure in terms of size and monomer incorporation is critical for polymeric biomaterials to move into the clinic. Two scaleable and reproducible processes to prepare amphiphilic terpolymers through a cationic polymerization for use in siRNA delivery are reported. The polymer-siRNA conjugates derived from polymers produced using these two methods are characterized in vivo, illustrating that control over the method of polymer synthesis can affect the polymer produced. Copyright

Full text of DOI:10.1002/pola.27094

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ARTICLE
Comparison of Flow and Batch Polymerization Processes for Production
of Vinyl Ether Terpolymers for Use in the Delivery of siRNA
1
2
3
3
2
Jason L. Nyrop, Arash Soheili, Rong Xiang, Fanyu Meng, Jacob H. Waldman,
3
4
4
4
Xiujuan Jia, Rubina Giarre Parmar, Benjamin W. Thuronyi, J. Michael Williams,
2
2
5
3
2
Lisa DiMichele, Michel Journet, Bonnie J. Howell, Bing Mao, Ian W. Davies,
4
5
2
Steven L. Colletti, Laura Sepp-Lorenzino, Erin N. Guidry
1
Department of Chemical Engineering, Merck & Co., Inc., Rahway, New Jersey 07065
Department of Process Chemistry, Merck & Co., Inc., Rahway, New Jersey 07065
Department of Analytical Chemistry, Merck & Co., Inc., Rahway, New Jersey 07065
Department of Medicinal Chemistry, Merck & Co., Inc., West Point, Pennsylvania 19486
Department of RNA Biology, Merck & Co., Inc., West Point, Pennsylvania 19486
2
3
4
5
Correspondence to: E. N. Guidry (E-mail: erin_guidry@merck.com)
Received 5 October 2013; accepted 9 December 2013; published online 5 February 2014
DOI: 10.1002/pola.27094
ABSTRACT: Synthetic polymers represent a modifiable class of
materials that can serve as adjuvants to address challenges in
numerous biomedical and medicinal chemistry applications
including the delivery of siRNA. Polymer-based therapeutics
offer unique challenges in both synthesis and characterization
as compared to small molecule therapeutics. The ability to con-
trol the structure of the polymer is critical in creating a thera-
peutic. Reported herein, are batch and flow polymerization
processes to produce amphiphilic terpolymers through a Lewis
ture in this rapid, exothermic, nonliving cationic polymeriza-
tion. In addition to analytical characterization of the polymers,
the in vivo activity of the polymer-siRNA conjugates is also
highlighted—demonstrating that the method of synthesis does
affect the in vivo activity of the resulting polymer conjugate.
V
C
2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym.
Chem. 2014, 52, 1119–1129
KEYWORDS: amphiphilies; biological applications of polymers;
cationic polymerization; copolymerization; drug delivery sys-
tems; poly(vinyl ethers)
3 2
acid BF OEt -catalyzed polymerization. These processes focus
on controlling reaction variables, which affect polymer struc-
INTRODUCTION Polymer-based delivery has received increas-
ing attention in the field of medicinal chemistry and biotech-
nology including the delivery of short interfering RNA
able analytical methods. For this reason, the process by which
the polymer is prepared defines the structure of the polymer
produced. As such, it is critical to develop reproducible and
scaleable polymerization processes that control variables, such
as temperature and mixing, which affect polymer molecular
weight, tacticity, and monomer incorporation. In this article,
we describe two methods, a modified batch process and flow
polymerization process, that control these variables, enabling
reproducible polymer synthesis. In addition to analytical char-
acterization of the polymers produced by these two methods,
the in vivo activity of their corresponding polymer conjugates
will also be reported; highlighting that in vivo activity is
affected by the polymerization method.
1–8
(siRNA).
The ability to control the character of the polymer
structure in terms of size and monomer incorporation pro-
vides a unique opportunity for chemists to optimize the deliv-
9–32
ery vehicle in concert with its payload.
Therefore, careful
control of the polymer synthesis is required and may provide
an opportunity to create a differentiated therapeutic. In a
recent report by Rozema et al., a poly(vinyl ether) (PVE)-based
cationic–amphiphilic polymer–siRNA conjugate was reported
to effect liver-targeted delivery of siRNA and produce mRNA
9
knockdown (KD) in a rodent model. The cationic–amphiphilic
PVE copolymer used to prepare the polymer–siRNA conjugate
was prepared through a nonliving BF OEt -catalyzed polymer-
EXPERIMENTAL
3
2
ization. While the molecular weight, polydispersity, and overall
monomer content can be measured for the terpolymer, little
direct structural information regarding the nature of monomer
incorporation or tacticity could readily be obtained by avail-
Materials
Unless otherwise noted, all chemicals were obtained from
the Aldrich Chemical Company. Carboxydimethylmaleic anhy-
dride poly(ethylene glycol) (CDM-PEG; M
5 680 g/mol)
w
Additional Supporting Information may be found in the online version of this article.
2014 Wiley Periodicals, Inc.
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and carboxydimethylmaleic anhydride N-acetylgalactosamine
neck round-bottomed flask with magnetic stir bar and inter-
nal thermocouple probe was set up in a heating mantle and
was fitted with a 14/20 Vigreux column (15 cm), distillation
head with water-cooled condenser, and collection flask in a
cold bath and purged with nitrogen for 15 min. The flask
was charged with BVE (1.50 L). The nitrogen line was moved
from the distillation pot to the collection flask and the pot
was heated to 100 C (internal temperature of 95 C) to
begin distillation into a dry ice/acetone-cooled collection
flask. Fractions (150–200 mL, colorless liquid) were col-
(
CDM-NAG) were synthesized according to the literature pro-
9
cedure. The siRNA targeting ApolipoproteinB using the Zim-
merman pattern was used.
3
8
0
0
5
3
-amil-GGAAUCUUAUAUUUGAUCCAsA-3
0
0
-UsCsCCUUAGAAUAUAAACUAGGUU-5
ꢀ
ꢀ
0
amil 5 amino linker; UC 5 2 -methoxy (OMe); AUG5 ribose;
s5 phosphorothioate linkage.
lected until <200 mL remained in the pot. Fractions were
1
The siRNA with an irrelevant sequence (Low Hex 9) was
used as a control.
checked by H NMR for purity (CDCl ). Fractions containing
3
large amounts of water, noted by ice crystals in the collec-
tion flask, were discarded.
0
0
0
5
3
-amil-iB-CUAGCUGGACACGUCGAUATsT-iB-3
Purification of Octadecyl Vinyl Ether
0
-UsUGAUCGACCUGUGCAGCUAU-5
Octadecyl vinyl ether (ODVE) was purchased from TCI. The
distillation apparatus was set up behind a blast shield. An
ammonia-rinsed 500-mL, three-neck round-bottomed flask
with large magnetic stir bar and internal thermocouple
probe was fitted with a 24/40 Vigreux column (30 cm) with
distillation head and air-cooled condenser. The flask was set
up in a heating mantle. The Coolant (water or air) in con-
denser should be warmed to room temperature to prevent
clogging of distillation equipment. ODVE (500 mL) was first
0
amil 5 amino linker; iB 5 Inverted deoxy abasic; CU 5 2 -
0
0
fluoro (F); AGT 5 2 -deoxy; UGA 5 2 -methoxy (OMe); AU5
ribose; s5 phosphorothioate linkage.
Synthesis of N-(2-Vinyloxy-ethyl)phthalimide Monomer
Potassium phthalimide (1400 g, 1 equiv.) was suspended in
DMF (5 L). The resulting mixture was heated to 93 C, and
then tetrabutylammonium bromide (48.67 g, 0.02 equiv.)
was added. The mixture was stirred for 0.5 h. The reaction
mixture was cooled to 60–70 C, and 2-chloroethyl vinyl
ether (1006.8 g, 1.25 equiv.) was added dropwise over 30
min. The reaction mixture was heated to 90–100 C for 1 h.
The mixture was cooled to 50–60 C, and then water (2.0 L)
ꢀ
ꢀ
warmed to 45 C to liquefy, and this was then charged to
ꢀ
the flask. The system was evacuated and purged with nitro-
gen three times to remove air (oxygen). The flask was evac-
ꢀ
ꢀ
uated (1 mmHg) and heated to 190–195 C (internal
ꢀ
ꢀ
temperature of 185–190 C). The flask was wrapped with
glass wool with aluminum foil outside that to contain heat
but the Vigreux column was left open to the air to create a
better temperature gradient. Fractions were collected until
was added dropwise over 1 h. The mixture was cooled to
ꢀ
20–30 C and aged for 30 min. The mixture was filtered
through a Celite pad, the solid was washed with water (2 3
L). The above solids were dissolved in CH Cl (7 L). The
<
100 mL remained in the pot. Each fraction was warmed to
1
2
2
ꢀ
40
C to melt before sampling for GC analysis. By GC, the
solution was concentrated to about 3–4 L, until solids
appeared. Cyclohexane (5 L) was added dropwise, and the
ratio of C18-vinyl ether:C16-vinyl ether >98:2 was achieved
after distillation.
ꢀ
mixture was further concentrated to 2–3 L at 45–50 C. The
ꢀ
mixture was cooled to 10–20 C and stirred for 30 min. The
Synthesis of Polymer 1
Synthesis of 18 kDa 15:4:1 [PiEVE:BVE:ODVE] by Flow
Polymerization
mixture was filtered and the cake was washed with cyclohex-
ꢀ
ane (2–3 L). The wet cake was dried under vacuum at 40 C
for 12 h. The desired product (PiEVE, 1.37 kg) was obtained
with 99.87% LCAP (0.13% of alcohol impurity) and 99.6%
LCWP in 83.2% corrected yield.
The monomer solution was prepared by dissolving ODVE
(
0.734 g, 2.5 mmol, 1 equiv.), n-BVE (0.99 g, 9.9 mmol, 4
equiv.), and N-(2-vinyloxy-ethyl)phthalimide (PiEVE; 8.03 g,
37 mmol, 15 equiv.) dichloromethane (150 mL) with a water
content of 100 ppm. The catalyst solution was prepared by
dissolving boron trifluoroetherate (0.107 g, 1.5 mol % vs.
monomers) dichloromethane (5.23 mL) with a water content
of 100 ppm. The quench solution was prepared by dissolving
2M ammonia in methanol (1.5 mL, 4 equiv. versus boron tri-
flouride diethyl etherate) dichloromethane (103 mL). Stream
1
H NMR (CDCl , 500 MHz): d 7.86 (dd, 2H, J 5 5.3, 3.0 Hz),
3
7
.72 (dd, 2H, J 5 5.4, 3.0 Hz), 6.42 (dd, 1H, J 5 14.4, 6.8
Hz), 4.20 (d, 1H, J 5 14.4, 2.2 Hz), 4.01 (m, 3H), 3.94 (m,
H). GC was used to measure residual solvent. The following
solvents were present in the following quantities: MeOH
100 ppm, DCM 540 ppm, cyclohexane 320 ppm, 2-
2
<
chloroethyl vinyl ether <100 ppm, and DMF <100 ppm.
00
1
is pumped at 1.429 mL/min through 1/16 PTFE and 316
Purification of Butyl Vinyl Ether
stainless steel tubing introduced to a controlled bath set at
ꢀ
n-Butyl vinyl ether (n-BVE) was purchased from Aldrich and
contained a stabilizer, 0.01% KOH. The peroxide content of
BVE was measured before distillation and was measured to
be <1 ppm (below limit of detection) using QuantofixPerox-
230 C. Stream 2 is pumped at 0.0714 mL/min through
0
0
1/16 PTFE and 316 stainless steel tubing introduced to the
controlled bath. Streams 1 and 2 mix in a 1-mm ID 316
00
stainless steel tee before entering a 30 mL coil of 1/8 304
stainless steel tubing. Stream 3 is pumped at 1.429 mL/min
ide 100 test strips. An NH -rinsed and oven-dried 2 L, three-
3
1
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0
0
through 1/16 PTFE and 316 stainless steel tubing introduced
to the controlled bath before mixing with the resulting stream
from the 30 mL coil (mixture of Streams 1 and 2) in a 1-mm
ID 316 stainless steel tee. The resulting stream exits the con-
trolled bath to a collection vessel. The collected polymer was
isolated by removal of dichloromethane under reduced pres-
sure to afford the product polymer 1 with a molecular weight
of Mw 5 18.1 kDa and polydispersity index of 1.7.
bath before mixing with the resulting stream from the 30
mL coil (mixture of Streams 1 and 2) in a 1-mm ID 316
stainless steel tee. The resulting stream exited the controlled
bath into a collection vessel. The collected polymer was iso-
lated by removal of solvent under reduced pressure to afford
the product polymer 3a with a molecular weight of Mw
5
27.1 kDa and polydispersity index of 2.2.
1
3
H NMR (500 mHz, CDCl ) 5 d 7.8–7.6 (om), 3.9–3.0 (om),
1
H NMR (500 mHz, CDCl ) 5 d 7.8-7.6 (om), 3.9–3.0 (om),
.9–1.1 (om), 1.0–0.7 (om).
1.9–1.1 (om), 1.0–0.7 (om). The same procedure was utilized
to synthesize two subsequent batches of polymer 3.
3
1
Synthesis of Polymer 2
Synthesis of 18 kDa 15:4:1 [PiEVE:BVE:ODVE] by Batch
Polymerization
Polymer 3b
1
M
w
5 29.4 kDa and polydispersity index of 2.0. H NMR
(500 mHz, CDCl ) 5 d 7.8–7.6 (om), 3.9–3.0 (om), 1.9–1.1
3
In a dry, three-neck flask fitted with an overhead stirrer, nitro-
gen inlet, and temperature probe was charged dichlorome-
(om), 1.0–0.7 (om).
Polymer 3c
thane (112 mL, <10 ppm H O). The flask was cooled to 240
2
1
ꢀ
Mw 5 28.0 kDa and polydispersity index of 2.8. H NMR
3 2
C (cryocool bath) then charged BF -Et O (1.19 mL, 9.44
(
500 mHz, CDCl ) 5 d 7.8–7.6 (om), 3.9–3.0 (om), 1.9–1.1
3
mmol). In a separate round bottom flask dissolved n-BVE
(
om), 1.0–0.7 (om).
(
(
48.9 mL, 378 mmol), ODVE (28 g, 94.0 mmol), and PiEVE
308 g, 1416 mmol) in dichloromethane (2100 mL, <10 ppm
Polymer Deprotection and Purification
H O). The solution of vinyl ether monomers was then charged
2
All polymers were deprotected and purified by the same
general procedure. The deprotection of polymer 2 is given as
an example. In a three-neck flask fitted with an overhead
stirrer, reflux condenser, and nitrogen inlet was slurried
polymer 2 (50.0 g, 79 mmol) in 2-propanol (1000 mL).
to the reaction vessel over 1 h at a constant rate using a Kna-
uer pump. The solution was then let stir for an additional 1 h
and then quenched with NH in MeOH (2.0M, 47 mL) and
3
removed from the cooling bath. Once at room temperature,
the reaction mixture was transferred to a recovery flask con-
centrated in vacuo to yield product polymer 2 (376 g, 598
mmol, 101% yield) as a foamy white solid with a molecular
weight of Mw 5 18.3 kDa and polydispersity index of 1.8.
Hydrazine (25% wt in H O) (499 mL, 3889 mmol) was
2
charged to the reaction vessel, and the reaction vessel was
ꢀ
heated (65 C). After 16 h, the reaction mixture was cooled
to room temperature. A constant volume distillation was per-
formed to remove 2-propanol while adding 0.1M NaOH to
maintain a volume of 1500 mL of total reaction volume. The
distillation was continued until the amount of 2-propanol
remaining in the reaction mixture was below 1% of the total
volume as monitored by GC. The aqueous polymer solution
was then subjected to tangential flow filtration (TFF) purifi-
1
H NMR (500 mHz, CDCl
.921.1 (om), 1.020.7 (om).
3
) 5 d 7.827.6 (om), 3.923.0 (om),
1
Synthesis of Polymer 3
Synthesis of 27.1 kDa 15:4:1 [PiEVE:BVE:ODVE] by Flow
Polymerization
The monomer solution was prepared by dissolving ODVE
cation (PALL centremate membrane, 1K
M
w
cutoff,
(6.31 g, 21.27 mmol, 1 equiv.), n-BVE (8.52 mL, 85.07 mmol,
OS001C12) with NaOH (0.25 N) until the high-performance
liquid chromatograph (HPLC) of the retentate polymer solu-
tion indicated complete removal of phthalhydrazide. The TFF
process was continued using water until the pH of waste
stream (permeate) became neutral (pH 7–8). The aqueous
solution was then freeze-dried to obtain the product polymer
4
equiv.), and PiEVE (69.30 g, 319.02 mmol, 15 equiv.) in
dichloromethane (900 mL) with a water content of 50 ppm.
The catalyst solution was prepared by dissolving boron tri-
fluoroetherate (0.92 g, 6.48 mmol, 1.5 mol % vs. monomers)
and dichloromethane (45 mL) with a water content of 50
ppm. The quench solution was prepared by combining 2M
ammonia in methanol (12.96 mL, 25.91 mmol, 4 equiv. vs.
boron triflouride diethyl etherate) was dissolved in dichloro-
methane (887 mL). To generate polymer, Stream 1 (mono-
mer solution) was pumped at 1.429 mL/min through 1/16
PTFE and 316 stainless steel tubing introduced into the con-
trolled temperature bath set at 230 C. Stream 2 (catalyst
solution) was pumped at 0.0714 mL/min through 1/16
2
-deprotected (20.3 g) as a sticky oil. The water content of
the isolated polymer was determined by thermogravimetric
analysis. The sodium content of the isolated polymer was
determined by inductively coupled plasma (ICP)-MS. The
weight percent of the isolated polymer was determined by
subtracting the weight of water and sodium hydroxide in the
polymer solid from the total weight of polymer solid.
0
0
ꢀ
0
0
1
PTFE and 316 stainless steel tubing introduced into the con-
trolled temperature bath. Streams 1 and 2 were mixed in a
H NMR (500 mHz, D O) 5 d 3.8–3.0 (om), 2.8–2.6 (om),
2
2.0–1.0 br, 0.9–0.7 (om).
1
-mm ID 316 stainless steel tee before entering a 30 mL coil
0
0
of 1/8 304 stainless steel tubing. Stream 3 (quench solu-
tion) was pumped at 1.429 mL/min through 1/16 PTFE
and 316 stainless steel tubing introduced to the controlled
Proton NMR
0
0
1
The H spectra were recorded on a Bruker AV or DPX series
NMR spectrometer at a frequency of 400 MHz or 500 MHz
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as noted and internally referenced to residual HOD at 4.80
vided as an example. Polymer 2-deprotected (1.2 g) was
placed in a 40 mL vial and was dissolved in 100 mM sterile
TRIS buffer at pH 9 (120 mL, 10 mg/mL) and added to a 1-
L sterile plastic bottle. To this solution was added SMPT
(Thermo Scientific) as 1 mg/mL solution in DMSO (18 mg,
1800 lL) corresponding to 1.5 wt % with respect to the
polymer weight. The solution was stirred for 1 h at rt to
generate activated polymer. The activated polymer solution
was further diluted using 100 mM sterile TRIS buffer at pH
9 (496 mL), followed by the addition of sata-modified siRNA
sata-ZimmApoB as a solution in water (250 mg, 32.4 mg/
mL, 7716 lL). This solution was aged for 4 h at room tem-
perature to generate the siRNA-polymer conjugate. In a sepa-
rate 1-L sterile plastic bottle, solid CDM-NAG (5.5 g) and
CDM-PEG (2.85 g) was added. The siRNA-polymer conjugate
solution was transferred by pouring into the plastic bottle
containing the CDM-NAG and CDM-PEG solids. The mixture
was stirred for 2 min to dissolve all solids and then trans-
ferred by pouring into the original plastic bottle, which con-
tained the siRNA-polymer conjugate. The reaction was
stirred for 1 h to generate the product masked siRNA-
polymer conjugate. The pH of the final solution was moni-
tored to ensure the pH was 8–9 throughout the conjugation
process. Purification of the masked polyconjugate was per-
formed using a TFF purification process. The amount of
siRNA covalently attached to PVE polymers (conjugation effi-
ciency) was determined using strong anion exchange chro-
matography (SAX). The conjugation efficiency of the product
2 2 3
ppm, CHD Cl at 5.32 ppm or CHCl at 7.27 ppm. Data for
1
H NMR are reported as follows: chemical shift (d), multiplic-
ity (s 5 singlet, d 5 doublet, t 5 triplet, q 5 quartet, m 5
multiplet, o5overlapped, br 5 broad multiplet) integration,
1
and coupling constant (Hz). H NMR spectra were in full
accordance with the expected structures.
Molecular Weight Determination Using Size Exclusion
Chromatography
Polymer molecular weight determination for protected polymers
was performed on an Agilent 1100 HPLC coupled with a Wyatt
TM
miniDAWN
TREOS [three-angle multiangle light scattering
(
MALS) system] and a Wyatt OptilabVR T-rEX (refractive index
detector). Chromatography was performed using two size exclu-
sion chromatographic columns in tandem, Waters Styragel HR3
Column, 5 lm, 7.8 3 300 mm (THF) and Waters Styragel HR4E
Column, 5 lm, 7.8 3 300 mm (THF) with 100% THF as mobile
phase at a flow rate of 1.0 mL/min. The temperature of the col-
ꢀ
umn was set at 25 C, and the UV detection wavelength was 260
nm. The polymer sample was dissolved in THF at 1–10 mg/mL
and 0.5 mg material was injected. Instrument normalization and
calibration was performed using 2–40K polystyrene standards
with a PDI of less than 1.1 (Polymer Laboratories). No calibration
standards were used in determination of molecular weights or
polydispersities. The dn/dc values were obtained for each injec-
tion assuming 100% mass elution from the columns. These val-
ues were also independently verified by measuring the dn/dc
independently using a Wyatt OptilabVR T-rEX refractometer. The
0
2a was measured as 89% by SAX (Table 3). The percentage
data was collected and processed using Wyatt Astra software.
Deprotected polymer molecular weight was not measured
directly. Deprotected molecular weight values were calculated by
adjusting the corresponding protected molecular weight based on
the mass loss due to removal of the protecting group.
of amines in a PVE polymer that are covalently modified
with disubstituted maleimides CDM-NAG and CDM-PEG
(molar basis) was determined by HPLC (masking efficiency).
0
The masking efficiency of the product 2a was measured as
0
50% (Table 3). The RNA concentration in the product 2a
was measured using ICP, and this concentration was used in
SATA Modification of Amino Zimmerman ApoB
Oligonucleotide
determining dilutions/dosage volumes for in vivo studies.
Amino Zimmerman ApoB Oligonucleotide
Amino zimmerman ApoB oligonucleotide (1 g, 0.0714 mmol)
was dissolved in 0.1 M sodium bicarbonate buffer (20 mL, 50
mg/mL) in a vial with magnetic stir bar and cooled to 0–5 C
in an ice water bath. In a separate vial SATA (83 mg, 0.357
mmol, 5 equiv.; Thermo Scientific part 26102) was dissolved in
Masked Polymer Conjugate Purification Process
A TFF process was used to purify masked polymer conjugate
0
formulations (i.e., 2a ) of unincorporated components and to
ꢀ
exchange buffer to a pharmaceutically acceptable formulation
vehicle. The TFF filter material was made of either modified
polyethersulfone (PES) or regenerated cellulose. The selection
of molecular weight cutoff for these membranes was done
with efficiency of purification and retention of polymer conju-
gate in mind. The processing parameters, including but not
limited to feed pressure, retentate pressure, crossflow rate,
and filtrate flux were set to allow reproducibility from batch
to batch and linear scaling of the process. Using the difiltration
mode of TFF, the reaction impurities were filtered out into the
permeate, while the retained polymer conjugate underwent a
buffer exchange. After TFF, the final product was concentrated
to 0.4–2.0 mg/mL of siRNA and sterile filtered using a 0.2-lm
0
.78 mL DMSO. The SATA solution was added over 1 min and
ꢀ
the clear, colorless reaction mixture stirred at 0–5 C for 2 h.
After 2 h, the reaction mixture was sampled and analyzed by
HPLC for consumption of the starting oligonucleotide. The reac-
tion mixture was purified by dialysis Millipore Amicon Ultra-15
Centrifugal Filter Unit with Ultracel-3 Membrane, UFC900324
using endonuclease free water until HPLC indicated the removal
of N-hydroxysuccinimide, and N-succinimidyl-S-acetylthioacetate.
The recovered solution was lyophilized to afford the product
SATA-ZimmApoB as a white fluffy solid. Sata Modified LH9 was
prepared by an identical procedure.
ꢀ
PES syringe filter and stored at 220 C until use.
Polymer Conjugate Synthesis
All polymer conjugates were prepared using the same gen-
eral procedure. Synthesis of polymer conjugate 2a is pro-
Conjugation Efficiency (siRNA-Polymer Conjugation)
SAX HPLC is used to determine the conjugation efficiency by
analyzing the final masked polyconjugate with and without
0
1
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dithiothreitol (DTT) treatment. Polymer conjugate solutions
were injected neat and as as a 1:1 mixture with 1.0M DTT
onto a Proteomix SAX-NP3 Column (Sepax Technologies, 3.0
are expressed as a ratio of ApoB mRNA/PPIB/GAPDH mRNA.
All mRNA data are expressed relative to the vehicle control.
2
lM, 100 3 4.6 mm ), with Mobile Phase A 5 100 mM TRIS
RESULTS AND DISCUSSION
(
pH 8.0), 10% ACN, and Mobile Phase B 5 100 mM TRIS
BF OEt2 Lewis acid-catalyzed cationic polymerizations of
vinyl ether monomers are known to be quite rapid and exo-
3
(pH 8.0), 10% ACN, 2M LiCl with a gradient of 0 to 100% B.
Free RNA duplex as well as free RNA duplex-dimer was
visualized using SAX chromatography. Total RNA (both free
and bound) was determined by using ICP spectroscopy. As
the RNA is the only phosphorus containing species in the
formulations, determining the total phosphorus content can
be used to directly determine the total RNA concentration.
Once the free RNA (duplex and duplex-dimer) and total RNA
is determined, the amount of RNA conjugated to the polymer
can be calculated (i.e., conjugation efficiency). Total RNA
thermic due to the conversion of pi-bonds to sigma-bonds
33,34
during chain propagation
making these types of poly-
merizations a challenge to control through a typical batch
polymerization due to poor heat dissipation and mass trans-
34–36
port.
From the literature, it is evident that a number of
variables, such as (1) monomer solubility, (2) temperature,
3) monomer concentration, (4) water content/monomer
(
purity, and (5) method of mixing/monomer addition, could
affect polymer structure and molecular weight in a nonliving
(bound and unbound RNA and RNA dimer) can also be
33,34
3 2
BF OEt -catalyzed cationic copolymerization (Fig. 1).
determined and visualized by pre-treatment of the polycon-
jugate with DTT before SAX chromatography. Conjugation
efficiencies are reported for all PVE polymer conjugates in
the text of the article.
While concentration, monomer solubility, water content, and
monomer purity could readily be addressed in a traditional
batch process, controlling temperature, and mixing is more
difficult given the rapid reaction kinetics. Two processes
were developed, a flow chemistry process and a modified
batch chemistry process, to address the poor heat dissipation
and mass transport issues associated with a traditional batch
process and allow for improved control of the resulting poly-
mer structure.
Masking Efficiency
Total concentrations of CDM-NAG and CDM-PEG were deter-
mined using reverse-phase HPLC (UV at a wavelength of 260
nm) with mobile phases of 0.1% TFA in water and 0.1% TFA
in 70/30 methanol:acetonitrile. Rapid demasking of the poly-
mer after injection onto the column allows quantitation of
CDMs with the polymer removed using a C18 guard column
to prevent chromatographic interference. Free (i.e., unbound)
CDM-NAG and CDM-PEG is analyzed by first filtering through
a 10 K centrifuge filter followed by analysis of the permeate
using the same reverse-phase HPLC method. Masking effi-
ciency can be calculated by first calculating the bound RNA,
CDM-NAG, and CDM-PEG. The polymer molecular weight in
combination with the total amines available for conjugation
is then used with the bound ligands to calculate masking
efficiency. Masking efficiencies are reported for all PVE poly-
mer conjugates in Table 3.
Flow Polymerization Process
BF OEt2 Lewis acid-catalyzed cationic polymerizations of
3
vinyl ether monomers are known to be quite rapid and exo-
thermic, and while this can make these types of polymeriza-
tions
a challenge to control through a typical batch
polymerization, these reaction characteristics made this poly-
merization an ideal candidate for a flow microreactor-based
3
5–37
approach.
The small dimensions of flow microreactors
enable extremely rapid mixing due to short diffusion path-
ways and the large surface-area-to-volume ratios allow for
efficient heat exchange properties.
The first step in optimization of the flow process was to
design a flow reactor that would control the reaction param-
eters, such as temperature and mixing, that had been identi-
fied as potentially affecting polymer structure and molecular
weight. In the design, a reaction stream containing a mixture
In Vivo Evaluation of Efficacy in Mice
CD1 mice were tail vein injected with the siRNA containing
polymer conjugates at a specified dose (mg/kg) in a volume
of 0.2 mL, 100 mM TRIS/9% glucose, pH9, vehicle. Forty-
eight hours post dose, mice were sacrifised and liver tissue
samples were immediately preserved in RNALater (Ambion).
Preserved liver tissue was homogenized and total RNA iso-
lated using a Qiagen bead mill and the Qiagen miRNA-Easy
RNA isolation kit following the manufacturer’s instructions.
Liver ApoB mRNA levels were determined by quantitative
RT-PCR. Message was amplified from purified RNA utilizing
primers against the mouse ApoB mRNA (Applied Biosystems
Cat. No. Mm01545156_m1). The PCR reaction was run on an
ABI 7500 instrument with a 96-well Fast Block. The ApoB
mRNA level is normalized to the housekeeping PPIB mRNA
and GAPDH. PPIB and GAPDH mRNA levels were determined
by RT-PCR using a commercial probe set (Applied Biosytems
Cat. No. Mm00478295_m1 and Mm4352339E_m1). Results
of the desired monomers was combined with the BF OEt2
3
catalyst solution by flowing these solutions into a mixing tee,
which results in very rapid and efficient mixing and rapid
heat dissipation. The tubing containing the monomer solu-
tion and catalyst solutions were immersed in a temperature-
controlled bath, which allowed for thermal equilibration of
the catalyst and monomer streams before their combination
in the mixing tee. After the monomer stream and catalyst
stream were combined in the first mixing tee, the resulting
crude polymerization stream was combined in a mixing tee
with a quench stream containing a solution of ammonia in
methanol. Again, the quench stream was immersed in a
temperature-controlled bath which allowed for thermal
equilibration of the quench solution before combining with
the polymerization stream (Fig. 1). The minimum residence
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FIGURE 1 (A) Synthesis of Lewis acid-catalyzed amphiphilic terpolymer. (B) Graphical representation of flow reactor used to pro-
duce polymers 1 and 3. (C) Graphical representation of batch polymerization reaction apparatus used to produce polymer 2.
time necessary for complete monomer consumption was
controlled by the length of the tubing between the first and
second mixing tee (i.e., the length of tubing between initial
mixing of the monomer and catalyst and the quench). Resi-
dence time was optimized experimentally by varying the tub-
ing length and monitoring for the presence of residual
monomer in the quenched reaction stream. Solvent water
content and temperature affect the polymer molecular
weight produced using the flow reactor. As can be seen in
Table 1, increasing water content decreases polymer molecu-
lar weight, and decreasing the reaction temperature
increases polymer molecular weight.
monomers was added very slowly to a solution of BF OEt2
3
at a rate such that mixing and reaction exotherm could be
adequately controlled (34.3 mmol/min, Fig. 1). No polymer-
ꢀ
ization was observed at temperatures below 260 C and so
ꢀ
240 C was selected as the lowest temperature for polymer-
ization evaluation. While it was not possible to reach molec-
ular weights as high as in the flow polymerization process
(max Mw 5 18 kDa vs. > 30 kDa), molecular weights could
still be controlled by solvent water content and reaction tem-
perature (Table 2).
In Vivo Comparison of Flow versus Modified Batch
Processes
Modified Batch Polymerization: An Inverse Monomer
Addition Process
In the traditional batch process used by Rozema et al.,
In order to examine the biological properties of PVEs pro-
duced using the modified batch and flow processes,
polymer-siRNA conjugates made from both processes were
prepared. The polymer conjugates, based on those reported
by Rozema consist of a polymer conjugated to siRNA through
a cytosolically labile disulfide bond. N-Acetylgalactosamine
(NAG), a hepatocyte-specific targeting ligand for the asialo-
gylcoprotein receptor, and PEG were conjugated to primary
amines of the polymer through an acid labile CDM linkage
9
BF OEt was added rapidly to a cooled solution of mono-
3
2
mers. While this approach does afford polymer, the reaction
is quite rapid and exothermic and reaction times of less than
1
minute for complete monomer consumption were
observed. In order to overcome the poor heat dissipation
and mixing observed in a traditional round bottom reaction
vessel, a batch process was developed in which a solution of
9
(Fig. 2). In order to eliminate molecular weight as a
1
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TABLE 1 Demonstration of the Relationship of Water Content
and Temperature to Polymer Molecular Weight Using the Flow
Polymerization Process
TABLE 2 Demonstration of the Relationship of Water Content
and Temperature to Polymer Molecular Weight Using the
Batch Polymerization Process
ꢀ
Water Content
ppm)
Temperature
M
w
(Protected)
Water Content (ppm) Temperature ( C)
w
M (Protected) kDa
ꢀ
(
( C)
kDa
1
1
3
6
0
0
5
0
240
235
235
212
18
16
10
8
5
1
2
5
5
1
5
5
2
0
230
230
230
220
220
220
210
210
210
29.5
17.3
7.8
00
00
24.3
18
All polymerizations were performed using
PiEVE:BVE:ODVE.
a 15:4:1 molar ratio of
0
00
12.1
19.4
13.4
6.1
percentage of total siRNA covalently attached to the polymer,
Table 3) and the CDM masking efficiency (percentage of total
amines in the polymer covalently modified with a CDM,
Table 3).
0
00
All polymerizations were performed using a 15:4:1 molar ratio of PiE-
VE:BVE:ODVE with a total monomer concentration of 0.45 M.
Polymer-siRNA conjugation efficiency was high (>85%) and
consistent across all polymer conjugates. CDM masking effi-
ciency was consistent across all samples (50–62%). CD1
mice were dosed i.v. with a solution of polymer conjugates
variable, conjugates were prepared using polymers of the
same molecular weight (Mw 5 18 kDa (protected)/Mw 5 9
kDa (deprotected), and using the same polymer:siRNA
weight ratio. Two RNA sequences were selected, ZimmApoB
which causes KD of ApolipoproteinB (ApoB), and a control
sequence Low Hex 9 (LH9), which does not result in KD of
any gene. Comparison of ApoB KD versus a control sequence
assures that KD is specific and not a result of toxicity of the
polymeric delivery vehicle. An identical process for generat-
ing polymer–siRNA conjugates was utilized for polymers
made through both the flow and modified batch processes.
0
0
1
a–c and 2a–c via tail vein injection. Livers were harvested
48-h postdose and assayed for apoB mRNA levels relative to
the mRNA levels of housekeeping genes PPIB and GAPDH
using RT-qPCR.
Robust mRNA KD was observed for all ApoB containing poly-
mer conjugates (1a–b and 2a–b , Fig. 3); whereas, very low
levels of KD were observed with the corresponding LH9 neg-
ative controls (1c and 2c ). However, polymer conjugates
prepared from polymers made using the flow process
(1a 2b ) were significantly more efficacious (95% KD) than
polymer conjugates prepared from polymers made using the
modified batch process (2a 2b , 50% KD) at the same dose
0
0
0
0
0
0
0
0
Polymer-siRNA conjugates (1a–c and 2a–c ) were character-
ized to determine the siRNA conjugation efficiency (molar
0
0
0
FIGURE 2 Graphical representation of dynamic polymer conjugate 1 . [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
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TABLE 3 Conjugation and Masking Efficiency Data for Polymer Conjugates Derived from Polymers Synthesized Using the Batch or
Flow Polymerization Processes
Polymer
Method of
Synthesis
RNA
Polymer:RNA
Conjugation
Masking
Conjugate
M
w
(Protected) kDa PDI Sequence wt:wt Ratio
Efficiency (%)
Efficiency (%)
0
1
1
1
2
2
2
a
Flow
Flow
Flow
Batch
Batch
Batch
18.1
18.1
18.1
18.3
18.3
18.3
1.7 ApoB
1.7 ApoB
1.7 LH9
4.8:1
11:1
11:1
4.8:1
11:1
11:1
87
91
85
89
92
90
62
51
55
50
55
53
0
b
0
c
0
a
1.8 ApoB
1.8 ApoB
1.8 LH9
0
b
0
c
FIGURE 3 Liver ApoB mRNA expression (mouse) at 3 mg/kg (48 h). [Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
(
3 mpk) and polymer:siRNA ratio (11:1). This difference in
amphiphilic terpolymers, it is necessary to use a mixture of
three vinyl ether monomers. From the literature, it is known
that steric and electronic factors can often affect vinyl ether
monomer reactivity and monomer reactivity will have a
direct impact on polymer structure in a copolymerization.
in vivo efficacy is suggestive that the method of synthesis of
PVE polymers affects the structure of the resulting polymer,
which in turn affects the in vivo properties of the resulting
polyconjugate.
From the in vivo results, it is clear that PVE copolymers with
the same Mw and overall monomer composition yield poly-
mer conjugates that have dramatically different in vivo per-
formance. In order to produce the desired cationic,
FIGURE 5 Size exclusion chromatographs for 18 kDa polymer
synthesized using either the flow polymerization (M
w
5 18.3
kDa, PDI 1.6) or batch addition processes (M 5 18.2 kDa, PDI
w
FIGURE 4 Plot showing average polymer composition versus
total monomer conversion for a flow polymerization with an
initial monomer charge of 15:4:1 PiEVE:BuVE:ODVE. [Color fig-
ure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
1.7). The solid lines are refractive index traces, red for flow poly-
mer; blue for batch addition polymer. The dotted lines are MALS
(multiangle light scattering) traces, red for flow polymer and
blue for batch addition polymer. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
1
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TABLE 4 Conjugation and Masking Efficiency Data for Polymer Conjugates Derived from Polymers Synthesized Using the Flow
Polymerization Process
Polymer
Method of
Synthesis
RNA
Polymer:RNA
Conjugation
Masking
Conjugate
M
w
(Protected) kDa PDI Sequence wt:wt Ratio
Efficiency (%)
Efficiency (%)
0
1
3
3
3
a
Flow
Flow
Flow
Flow
18.1
27.1
29.4
28.0
1.7 ApoB
2.2 ApoB
2.0 ApoB
2.8 ApoB
4.8:1
4.8:1
4.8:1
4.8:1
87
92
85
89
62
50
55
53
0
a
0
b
0
c
The composition of a copolymer cannot be determined from
knowledge of the homopolymerization rates of the mono-
mers, as the relative rates of monomer copolymerization
often bear little resemblance to the relative rates of homopo-
Given the nonliving nature of the polymerization, it is impossi-
ble to unambiguously define the polymer structure (Fig. 5).
However, we speculate that polymers produced by a flow poly-
merization process have a more gradient-type structure while
polymers produced by a modified batch process have mono-
mers distributed more evenly throughout the polymer chain.
3
3,34
lymerization.
In order to understand monomer reactivity
in the terpolymerization, the copolymerization must be
quenched at various levels of monomer conversion. In the
modified batch polymerization process, the reaction was so
rapid that it was not possible to quench the reaction quickly
enough to get a range of different levels of monomer con-
sumption. However, manipulating reaction time in the flow
polymerization process was quite straightforward and could
be readily controlled by manipulation of the reaction resi-
dence time by varying the length of tubing between the ini-
tial mixing tee and the quenching mixing tee. In this way, it
was possible to get a snapshot of the unconsumed monomer
ratio at various levels of monomer conversion. From a series
of quenching experiments, it was determined that phthalim-
ide ethyl vinyl ether (PiEVE) is less reactive than the alkyl
vinyl ethers (BVE, ODVE, Fig. 4). This differential reactivity
leads to an average polymer composition that changes over
time. The relative amount of PiEVE in the terpolymer at low
conversion is lower than that contained at higher levels of
conversion. Given the monomer reactivity data and differ-
ence in experimental setup, we anticipate that polymers pro-
duced by different methods may have a different distribution
of monomers along the polymer chain. In the flow polymer-
ization process, the catalyst and bulk monomer are intro-
duced to each other simultaneously. In the batch
polymerization process, monomer is being continuously
added to a system that already contains growing polymer
chains.
Flow Polymerization: Batch-to-Batch Comparison
Superior gene silencing was observed for polymer–siRNA
conjugates made with polymers synthesized using the flow
polymerization process. In order to demonstrate that the
flow process was not only capable of producing polymer–
siRNA conjugates that effect robust gene silencing but also
of producing polymer–siRNA conjugates that reproducibly
have the same biological activity as measured by in vivo
mRNA KD, multiple batches of polymer were produced using
the flow process. After deprotection, each polymer was con-
jugated to ApoB siRNA using an identical conjugation pro-
cess. The masking efficiencies and siRNA conjugation
efficiencies were consistent across all three polymer conju-
gates (Table 4). CD1 mice were dosed i.v. at 1 mpk with a
0
solution of polymer conjugates 3a–c via tail vein injection.
FIGURE 7 MALDI-TOF comparing protected polymers of the
same average molecular weight (18 kDa) generated using the
flow. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
FIGURE 6 Liver ApoB mRNA expression (mouse) at 1 mg/kg
(
48 h). [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
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Livers were harvested 48-h postdose and assayed for apoB
mRNA levels relative to the mRNA levels of housekeeping
genes PPIB and GAPDH using RT-qPCR. Consistent mRNA KD
was observed throughout the three batches of flow-based
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Fig. 5). Polymer (1–5 mg/mL) in THF was mixed with the
matrix solution (10 mg/mL sinapinic acid in THF) at a 1:1 ratio.
The resulting mixtures were then deposited on the MALDI tar-
get. Each sample was analyzed twice to show the reproducibil-
ity of the analysis process. A Bruker Autoflex III mass
spectrometer was used to analyze the samples in the positive,
lineal mode. The resulting MS data (Fig. 7) were smoothed (Sav-
tizkyGolay, 0.2 m/z width, one cycle) and baseline substracted
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were observed for these two different polymer samples. Indeed,
within this region, the major series of the MALDI signals have a
difference of 100 Da, a likely indication of the incorporation of
an extra BVE unit. This data is consistent with the method of
synthesis affecting the composition of the copolymer.
1
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We have developed two scaleable and reproducible proc-
2
2 R. G. Parmar, M. Busuek, E. S. Walsh, K. R. Leander, B. J.
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3
2
Howell, L. Sepp-Lorenzino, E. Kemp, L. S. Crocker, A. Leone, C.
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catalyzed polymerization. In addition to traditional analytical
characterization, the polymer–siRNA conjugates derived from
polymers produced using these two methods were character-
ized in vivo. Polymers made through the flow polymerization
process were shown to be more efficacious when formulated
as siRNA–polymer conjugates than those made through the
modified batch process. This work illustrates that control
over the method of polymer synthesis can affect the polymer
produced, thus affecting the in vivo performance.
23 S. E. Barrett, R. M. Garbaccio, R. A. Kowtoniuk, W. Wang, J.
M. Williams, J. Liao, T. Pei, S. C. Tobias, 2013, PCT Int. Appl.
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5 S. E. Barrett, M. Busuek, S. L. Colletti, R. M. Garbaccio, E. N.
Guidry, R. A. Kowtoniuk, J. Liao, C. A. Parish, R. G. Parmar, T.
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ACKNOWLEDGMENTS
The authors thank Enrique Vazquez for providing siRNA
materials.
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