Journal of Pharmaceutical Sciences 111 (2022) 2639−2644

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Journal of Pharmaceutical Sciences

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

Note

Impact of Annealing and Controlled Ice Nucleation on Properties

of A Lyophilized 50 mg/ml MAB Formulation
Jijun Wanga,*, James A. Searlesa, Ekaterina Torresa, Serguei A. Tchessalovb,
Anthony L. Younga
a
Pharmaceutical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc, Chesterfield, MO 63017, USA
b
Pharmaceutical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc, Andover, MA 01810, USA

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

Article history: We compared “ice-fog” controlled ice nucleation at -6 °C to annealing at the same temperature for a
Received 7 January 2022 50 mg/mL monoclonal antibody formulation, using shelf-ramp freezing as a control. Cake structure, drying
Revised 18 May 2022 time, reconstitution time, specific surface area, calculated cake resistance and size exclusion chromatography
Accepted 18 May 2022
were all compared. Controlled nucleation resulted in the fastest reconstitution, shortest primary drying, low-
Available online 22 May 2022
est calculated cake resistance, lowest specific surface area and highest moisture content. There was no effect
upon the results for size exclusion chromatography. Results for annealing were between those for controlled
Keywords:
nucleation and shelf-ramp freezing. All results were consistent with “ice-fog” controlled nucleation at -6 °C
Freeze-drying
Lyophilization
having greater impact upon the ice crystal morphology than annealing at the same temperature for 3 h.
Protein © 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open
Monoclonal antibody access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Morphology
Nucleation
Protein formulation(s)
Drying
Solid-state
Amorphism

Introduction
Lyophilization is a well-established drying process for stabilization
of biologics.1-4 There are three major steps in the lyophilization pro-
cess: freezing (solidification) (may or may not include an annealing
step), primary drying (ice sublimation), and secondary drying (mois-
ture desorption).1,5 The structure of the dry layer above the ice inter-
face determines the cake resistance to water vapor flow during
primary drying, and hence the required primary drying time. This
structure, while it can be affected during drying by temperature
excursions above the collapse temperature, is initially set by the freez-
ing and annealing steps.6 The final cake structure has a strong influ-
ence upon reconstitution time and other product quality attributes.
Annealing is frequently used after shelf-ramp freezing to reduce
product resistance during primary drying, resulting in a higher pri-
mary drying rate.7-9 However, annealing also causes reduction of the
product specific surface area, which can decrease the water desorp-
tion rate in secondary drying and may lead to increased residual
moisture content in the final product.4,10
* Correspondence author.
There are several reports that show annealing can reduce recon-
stitution time when annealing results in crystallization of formula-
tion components such as mannitol.11,12 Annealing as also been
shown to affect reconstitution time for amorphous protein formula-
tions similar to what we used.13-16 Blue et al. showed that incorporat-
ing an annealing step after freezing can reduce the reconstitution
time for two separate mAbs by up to 60%.13
Controlled ice nucleation (CN) is receiving consistent attention
due to its benefits in terms of improving lyophilization process per-
formance and enhancing product quality attributes of lyophilized
drug products. Several controlled ice nucleation systems have been
developed and are commercially available, such as ice-fog, pressuri-
zation/depressurization, vacuum-induced, or partial vacuum.17,18
Several papers have reported that controlled ice nucleation can
also reduce the reconstitution time of high concentration protein
formulations.12,16,19,20 Geidobler et al. found that CN at -5 °C led to a
66% reduction in reconstitution time of a lyophilized mAb at
161 mg/mL.19 About 50% reduction was reported by Singh et al. for a
mAb at 108 mg/mL.20
In contrast to others, Luoma et al. compared different CN techni-
ques with 10 mg/mL and 100 mg/mL mAb formulations and found
that the partial vacuum and depressurization techniques actually

https://doi.org/10.1016/j.xphs.2022.05.016
0022-3549/© 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
2640 J. Wang et al. / Journal of Pharmaceutical Sciences 111 (2022) 2639−2644
increased reconstitution time, and that ice-fog CN produced results
similar to shelf-ramp freezing.18
There is a lack of published data comparing annealing and con-
trolled nucleation in which both processes are carried out at the same
temperature. We compared controlled ice nucleation at a shelf tem-
perature of -6 °C with annealing at same temperature (-6 °C), evaluat-
ing cake structure, primary drying time, reconstitution time, specific
surface area, and size exclusion chromatography. We saw improve-
ments in lyophilization cycle time and in product reconstitution time
with controlled nucleation and found that similar benefit obtained by
using controlled nucleation could also be achieved using annealing.
Materials and Methods
Materials
The protein used in this study was a IgG2 monoclonal antibody
(mAb) manufactured by Pfizer. The mAb formulation was 50 mg/mL
IgG2 in 10 mM histidine buffer pH 5.5 containing 8% sucrose and
0.2 mg/mL polysorbate-20. The excipients chosen for the studies were
based on those typically used in protein formulations.21 The mAb bulk
formulation was filtered through a 0.22 mm membrane filter and was
filled into 10 mL Type I clear glass vials with 4 mL fill volume in a gen-
eral laboratory environment. The vials were partially stoppered with
20 mm FlurotecÒ coated butyl rubber stoppers for lyophilization.
Histidine was purchased from JT Baker (Center Valley, PA). Poly-
sorbate-20 (containing low carbonyl and peroxide) was purchased
from Thermo Scientific (Rockford, IL). Sucrose was obtained from
Pfanstiehl Laboratories (Waukegan, IL). EDTA was from JT Baker (Cen-
ter Valley, PA). Distilled, deionized water was used throughout.
Lyophilization and Controlled Nucleation (CN)
All samples were freeze-dried using a SP LyoStar-3 lab scale
freeze-dryer (SP Scientific, Stone Ridge, NY). One shelf was loaded
with 30-50 samples surrounded by placebo vials to completely fill
the shelf. Product temperature was measured using thermocouples
inserted into sample vials. Table 1 shows the freezing and drying con-
ditions used for the three different cycles tested.
The controlled nucleation and annealing temperature of -6 °C (shelf
temperature) was selected to be far enough below zero to give confi-
dence that even the edge vials on the shelf would not melt completely.
For shelf ramp freezing without annealing, the shelves were at 20 °C
for loading, the chamber was ramped at 1 °C/min to 5 8C and then
ramped at 0.5 °C/min to -45 8C and holding for 90 minutes. The cycle
named “Annealed -6 8C” samples were loaded on the shelf pre-cooled
to 5 8C, held for 10 minutes, then the shelf temperature was ramped
down to -45 °C at 0.5 8C/min then held for 90 minutes. For annealing
the shelf was ramped up to -6 8C at 0.5 8C/min, held for 3 h, re-cooled
to -45 8C at 0.5 8C/min, and held for 90 minutes to re-freeze. The
Table 1
Lyophilization cycle parameters. All shelf temperature ramp rates up and down were 0.5 °C/min except where noted. All temperatures are shelf fluid inlet temperature.
Shelf Ramp Freezing
Sample loading 20 °C
Freezing Ramp 20 °C to 5 °C at 1 °C/min then
to -45 °C at 0.5 °C/min, hold 90 min
Annealing n/a
Drying pressure
Primary drying
Secondary drying
Total cycle time (includes 110 h
annealing and CN equilibration time)
“Controlled nucleation -6 8C” cycle used a VERISEQÒ ice-fog nucleation
system (IMA Life/Linde, Tonawanda, NY). For this process, the sample
vials were loaded and held on the -6 8C shelf for 2.5 h, after which the
chamber pressure was reduced to 2.8 psia and nitrogen gas carrying
ice crystals was introduced into the chamber to induce nucleation. The
shelf temperature was then ramped to -45 8C at 0.58C/min.
Primary drying conditions were set to maintain the product tem-
perature near or below its collapse temperature while in the presence
of ice, which was determined to be approximately -28 8C using
freeze-drying microscopy. At the end of freeze drying, the product
chamber was backfilled with nitrogen to approximately 600 Torr and
the vials were stoppered before unloading. Samples were crimped to
seal with flip-off seals and kept refrigerated until analysis.
Moisture Determination
Karl Fischer (Mettler Toledo, Columbus, OH) was used to determine
residual water content in the lyophilized cake. The titration sample was
prepared by dissolving/suspending the cake in a diluent of 50% formam-
ide/ 50% methanol mixture. The samples were then vortexed to extract
residual water before the coulometric titration for water content.
Scanning Electron Microscopy (SEM)
Lyophilized cake samples were mounted on Scotch tape-covered
stubs using silver paste. Samples were sputter-coated with 10 mm gold
using Gressington 108 instrument (Gressington Scientific Instruments,
Watford, UK) prior to imaging. Gold coated samples were analyzed using
a JOEL JCM-600-Plus scanning electron microscope (JOEL, Peabody, MA)
using secondary electron detector at 5 keV and 200-300 mBar.
Specific Surface Area (SSA)
Gemini 2380 instrument (Micromeritics, Norcross, GA) was used
to measure the SSA of the freeze-dried samples. Samples were placed
in the bulb glass tubes in a dry box and passively purged with dry
nitrogen for at least 48 h. The instrument provides an option to use
free space balancing with the glass beads and this approach makes
nitrogen gas acceptable for measurement of low SSA. Sample and ref-
erence tubes were evacuated at 200 mmHg/min. SSA of each sample
was measured using Nitrogen with multi-point BET analysis method.
Free space differential was measured using helium. To facilitate
measurements with the low SSA tube was partially filled with glass
beads to minimize differences in the free space volume. The weight
of samples was used to calculate the specific surface area
Reconstitution Time
The lyophilized cakes were reconstituted by addition of 3.6 mL
water for injection (WFI). Reconstitution was performed by applying
Annealed -6 °C Controlled Nucleation -6 °C
5 °C -6 °C
Ramp 5 °C to -45 °C, hold 90 min Hold -6 °C for 150 min, ice fog controlled
nucleation, ramp to -45 C and hold 90 min
Ramp -45 °C to -6°C, hold 180 min, n/a
ramp -6 to -45 °C, hold 90 min
50 mTorr
Ramp − 45 °C to -25 °C, hold 90 h
Ramp -25 °C to +35 °C at 0.2 °C/min, hold 10 h
116 h 110 h
 J. Wang et al. / Journal of Pharmaceutical Sciences 111 (2022) 2639−2644 2641

gentle swirling after the addition of water. Reconstitution time was monomeric species is identified by its characteristic retention time
determined by visual observation of complete dissolution. The recon- relative to a reference standard. Typical method precision was
stitution time was determined with three cake samples and reported approximately 0.1% RSD for IgG monomer.
with an average of the three measurements.

Results and Discussion

Size Exclusion Chromatography (SEC)
Separation was performed with YMC-Pack Diol-200, 300 x 8 col-
umns, neutral phosphate buffer/NaCl mobile phase, and detection at
280 nm sing Agilent HPLC system (Agilent, Santa Clara, CA). The
The lyophilization cycle run profiles for the three cycles are shown
in Fig. 1a-c, and plots of freezing are in Fig. 1d-f. Primary drying
times, shown in Table 2, were calculated as the time for the Pirani
pressure sensor to equilibrate close to the pressure measured by the

Figure 1. Lyophilization parameter profiles: (a-c) entire cycle (green: pirani pressure, red: CM pressure, blue: CM temp, all other color: probe temp); (d-f) freezing; (g) cake resis-
tance. Runs: (a and d) shelf-ramp frozen, no annealing; (b and e) annealed; (c and f) controlled nucleation.
2642 J. Wang et al. / Journal of Pharmaceutical Sciences 111 (2022) 2639−2644
Table 2
Lyophilization and product quality results.
Sample Primary Drying Time (hr) Moisture (%)
Shelf-Ramp Freezing 87 0.2
Annealed -6 °C 62 0.5
Controlled Nucleation -6 °C 61 0.9
capacitance manometer. Primary drying time was significantly
reduced (30% reduction) by CN compared to shelf-ramp freezing
without annealing. This result agrees with other reported CN
studies. For example, Awotwe-Otoo et al compared conventional
shelf-ramp freezing to pressurization/ depressurization CN, and
primary drying time was reduced by 19%.22 A similar study
reported by Konstantinidis et al achieved a 41% reduction in pri-
mary drying using CN.23 In comparison to the 30% decrease in
primary drying that we observed with CN at -6 °C, we found a
nearly identical reduction of 29% with simple annealing at -6 °C.
Note that residence time of product between -6°C and 0°C
(Fig. 1b), after nucleation was induced, was about 1 h as opposed
to 3 h during annealing step.
Cake resistance to the flow of escaping water vapor is an impor-
tant factor used to characterize how ice crystal size impacts product
temperature and drying rate during lyophilization.22 Fig. 1g shows
calculated cake resistance curves for the three cycles used in this
study. The resistance of cake as a function of dry layer was calculated
from the product temperature profile, using the approach used by
Tchessalov et al.24 The cake resistance for the controlled nucleation
and annealed cycles were much lower than the shelf-ramp frozen
cycle. For parts of the cycle the annealed product had slightly higher
resistance than the CN product. Our findings are similar as reported
by Gieseler who found that cake resistance after controlled nucle-
ation freezing at -3 °C was reduced by about 25% compared to shelf
ramp freezing. Annealing at -15 °C for 6 h resulted in cake resistance
similar to CN.25
Figure 2. Cake Appearance: self ramp frozen (A), annealed -6 °C (B), and controlled nucleation (C) cycles.
Recon. Time (sec) Surface Area Multi-point BET (m2/g) HMMS (%) Monomer (%)
129 § 11 1.4 1.9 98.1
71 § 6 0.8 2.0 98.0
49 § 4 0.6 1.9 98.1
The lyophilized cakes from the three cycles each appear slightly
different. As seen in Fig. 2, the CN cake appears less elegant due to
larger pores and some cracks. Microscopic appearance in Fig. 3 con-
firms the larger pores of the CN cake, especially at the bottom of the
cake. The SEM images in Fig. 3 also show that pore sizes at the bottom
of the cakes are progressively larger in the order shelf ramp frozen <
annealed < CN. Our findings in this regard are not unique. Gieseler
reported the cake with CN had a more porous and fragile cracked
structure.25
SSA of the samples were 1.4 m2/g for shelf-ramp frozen without
annealing, 0.8 m2/g for annealed, and 0.6 m2/g for controlled nucle-
ation. These results align with the other observations given that the
SSA of the dried cake is initially established by the freezing / anneal-
ing process (as long as the lyophilization cycle is sufficiently conser-
vative to avoid collapse). The larger SSA of the product prepared with
CN method agrees with other reports.20, 22 Awotwe-Otoo et al
reported that CN resulted in cake of a mAB formulation with SSA of
0.46 m2/g compared to shelf-ramp frozen SSA of 0.90 m2/g.22
As discussed above, others have found that CN could reduce
reconstitution time of lyophilized products.19,22 We observed a simi-
lar result in which the reconstitution time of the CN cake was
reduced to 49 seconds from 129 seconds (Table 2). In contrast, the
reconstitution time of annealed samples was 71 seconds, slightly lon-
ger than CN but still faster than the controls. This result agreed with
the results reported by others.13,15,16 The shorter reconstitution times
for the CN and annealed cakes were likely due to the more open net-
work of larger pores.15
 J. Wang et al. / Journal of Pharmaceutical Sciences 111 (2022) 2639−2644
Figure 3. SEM of lyo cake: Shelf ramp frozen (A), annealed -6C (B), and controlled nucleation (C) cycles. Top images are from the top of the cakes and bottom images are from the
bottom of the cakes.
The residual moisture contents (Table 2) were in order: 0.2% for
shelf-ramp frozen without annealing, 0.5% for annealed, and 0.9% for
CN. Higher moisture for CN has been reported in many
publications5,19,22,26 and is consistent with the SSA differences.
Higher SSA for shelf-ramp freezing product is responsible for its
lower moisture content. The higher moisture level in the annealed
cake observed in this study indicated that the -6 °C annealing also
increased the crystal size and decreased the water desorption rate in
secondary drying, which was similar to what was observed for the
CN cycle.
Protein aggregates can occur during formulation, lyophilization
processes and storage.27 To assess the quality of the lyophilized cake
produced from shelf-ramp frozen, annealed, and CN cycles, soluble
aggregates of the protein in reconstituted solutions were determined
using the SEC method (Table 2). There was no significant change in
high molecular weight species or % monomer for all samples regard-
less of lyophilization conditions. Fang et al reported that lyophilized
IgG and rHSA proteins with lower SSA can reduce aggregation28 due
to the reduction of adsorption of protein at the ice/freeze-concentrate
interface.29 We did not see a difference in aggregation among the
controlled and non-controlled nucleation methods. This could be due
to the presence of polysorbate-20 in the formulation, the purpose of
which is to reduce aggregation, as well as the relatively high protein
concentration of 50 mg/mL.
Conclusions
This study compared controlled ice nucleation and traditional
annealing processes, in which both were carried out at -6 °C. Primary
drying time, cake reconstitution time, specific surface area, crystal
and pore size, SEC and calculated dry layer cake resistance were eval-
uated. The results show that while “ice-fog” CN at -6 8C was most
effective at reducing primary drying time, cake resistance, specific
surface area, and reconstitution time; annealing at -6 8C after shelf-
ramp freezing yielded results that were nearly as good. As expected,
2643
moisture content trended inversely with these factors, meaning that
shorter primary drying would be at least partially offset by additional
secondary drying time and/or higher secondary drying temperature.
Declaration of Competing Interests
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.
Acknowledgements
The authors thank Bakul Bhatnagar for his valuable conversations
in the experiment design and tests. This research did not receive any
specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
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