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Vacuum-Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How Does Nucleation Control Affect Protein Stability?

April 2019 · Journal of Pharmaceutical Sciences 109(1)

DOI:10.1016/j.xphs.2019.04.014
License · CC BY-NC-ND 4.0

Authors:

Irene Oddone Andrea Arsiccio

Chinwe Duru Kiran Malik
Politecnico di Torino Coriolis Pharma

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Citations (11) References (56) Figures (5)

Abstract and Figures

In the present work, the effect of controlled nucleation on the stability of human growth
hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum- Discover the world's
research
induced surface freezing technique has been compared to conventional freezing, both with
and without an annealing step. Size exclusion chromatography and cell-based potency 25+ million
assays have been used to characterize the formation of soluble aggregates and the members
biological activity of hGH, respectively. The results obtained indicate that controlled
nucleation has a positive effect onDiscover the
both cycle world's research
performance and product homogeneity 160+ million
because of the formation of bigger ice crystals, and characterized by a narrower publication
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dimensional distribution. From the point of view of hGH stability, we observed that vacuum-
induced surface freezing is not detrimental to the biological activity of the protein, or 2.3+ billion
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aggregate formation. In addition, the effect of 2 different formulations, including trehalose citations
or cellobiose, on protein preservation was also considered for this study. Join for free

SEM images of Percentage of Representative Drying Time Pore Dimension

the samples… dimers and hig… dose-response… (Difference… D p in the Dried…

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 Journal of Pharmaceutical Sciences 109 (2020) 254-263

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences

j o u r n a l h o m e p a g e : w w w. j p h a r m s c i . o rg

Pharmaceutical Biotechnology

Vacuum-Induced Surface Freezing for the Freeze-Drying of the

Human Growth Hormone: How Does Nucleation Control Affect
Protein Stability?
Irene Oddone 1, Andrea Arsiccio 1, Chinwe Duru 2 , Kiran Malik 2 , Jackie Ferguson 3 ,
Roberto Pisano 1, Paul Matejtschuk 2 , *
1
Department of Applied Science and Technology, Politecnico di Torino, 24 corso Duca degli Abruzzi, Torino 10129, Italy
2
Standardisation Science Section, Analytical and Biological Sciences Division, National Institute for Biological Standards and Control, Blanche Lane,
Medicines & Healthcare Products Agency, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
3
Biotherapeutics Division, National Institute for Biological Standards and Control, Medicines & Healthcare Products Agency, Blanche Lane, South
Mimms, Potters Bar, Hertfordshire EN6 3QG, UK

a r t i c l e i n f o a b s t r a c t

Article history: In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH)
Received 31 January 2019 during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing
Revised 6 April 2019 technique has been compared to conventional freezing, both with and without an annealing step. Size
Accepted 9 April 2019
exclusion chromatography and cell-based potency assays have been used to characterize the formation of
Available online 17 April 2019
soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that
controlled nucleation has a positive effect on both cycle performance and product homogeneity because
Keywords:
freeze-drying
of the formation of bigger ice crystals, an d characterized by a narrower dimensional distr ibution. From
nucleation the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental
protein aggregation to the biological activity of the protein, or aggregate formation . In addition, the effect of 2 different
protein formulation
formulations, including trehalose or cellobiose, on protein preservation was also considered for this
HPLC
biopharmaceutical characterization study.
Crown Copyright © 2020 Published by Elsevier Inc. on behalf of the American Pharm acists Association.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

Introduction phases can be carried out at lower temperature with respect to

other drying processes, thus preventing damage to heat-sensitive
Freeze-drying is the most commonly used method for the molecules. In spite of this, the freeze-drying process generates
preparation of solid protein-based pharmaceuticals. During a several stresses, which could lead to loss of activity of
freeze-drying cycle, temperature is lowered so as to freeze the biopharmaceuticals. 1
product; subsequently, 2 drying steps, called primary and second- Many of these stresses are linked to the freezing step. Cold
ary drying, are performed, in which water is removed at low denaturation of the protein could arise as a consequence of the low
pressure by sublimation or desorption, respectively. The 2 drying temperature used during freezing, 2 and the active molecule may
3
also adsorb onto and unfold at the ice-water interface. The exis-
tence of surface-induced denaturation phenomena is demonstrated
by the smaller recovery of protein usually observed at high cooling
Abbreviations used: C þ M, cellobiose þ mannitol formulation; DMA, dynamic rates. 3,4 This is due to the smaller dimension, and, therefore, greater
mechanical analysis; FDM, freeze-drying microscopy; GMP, Good Manufacturing
surface area to volume ratio, of ice crystals formed at high cooling
Practice; hGH, human Growth Hormone; HPLC-SEC, high performance liquid size
exclusion chromatography; LDH, lactate dehydrogenase; MDSC, modulated differ- rates. Another clear evidence of this phenomenon is the decrease in
ential scanning calorimetry; SEM, scanning electron microscopy; T þ M, the percentage of unfolded protein molecules observed by
Trehalose þ Mannitol formulation; VIN, vacuum induced nucleation; VISF, vacuum increasing the protein concentration in formulations to be frozen,
induced surface freezing; XRD, X-ray diffractometry.
which suggests that protein denaturation is limited by the finite
* Correspondence to: Paul Matejtschuk (Telephone: 0 (þ 44) 1707 641515). 5
E-mail address: [email protected] (P. Matejtschuk).
extension of the ice-freeze concentrate interface.

https://doi.org/10.1016/j.xphs.2019.04.014
0022-3549/Crown Copyright © 2020 Published by Elsevier Inc. on behalf of the 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/).
 I. Oddone et al. / Journal of Pharmaceutical Sciences 109 (2020) 254-263 255

Moreover, some components of the formulation could phase competitive with other controlled freezing strategies. Moreover, an
separate during freezing, thus providing another interface that important advantage of the VISF technique is the possibility to
6
could generate surface-induced denaturation. In addition to this, precisely control nucleation temperature, without requiring any
some of the most commonly used buffers, such as the phosphate additional equipment.
buffer, could precipitate, leading to a wide pH shift that can dena- Oddone et al. 23,24 showed that VIN can be highly beneficial for
ture the protein to a varying degree. 7,8 Finally, cryoconcentration is homogeneity of the batch and optimization of the freeze-drying
another possible source of stresses for the biopharmaceuticals to be cycle. However, the impact of controlled nucleation on the stabil-
freeze-dried. The rapid increase in solutes concentration, combined ity of biopharmaceuticals requires further investigation. Geidobler
to selective crystallization of formulation components, may lead to et al.26 evidenced that controlled ice nucleation may be applied to
significant changes in ionic strength and relative composition of the reduce reconstitution time of highly concentrated lyophilized
9
amorphous phase, possibly destabilizing a protein. As a result of protein product, and it was reported that it also helped to suppress
the increased solute concentration, chemical reactions may actually glass fogging, that is, the undesired migration of protein solutions
accelerate in a partially frozen aqueous solution. 10 However, it was up on the inner walls of glass vials during the freezing step of
shown that the critical destabilizing factor is related to adsorption lyophilization. 27 Moreover, Fang et al. 28 studied the effect of
at the ice-freeze concentrate interface. 11 It is therefore clear that a controlled nucleation on the stability of lactate dehydrogenase
proper control of freezing is of utmost importance because it could (LDH) during both freezing and freeze-drying, but their results
lead to improved recovery and increased stability of the therapeutic were not conclusive. In fact, they found that controlled nucleation
protein being processed. remarkably improved LDH recovery after freeze-thawing and also
An event which strongly affects the outcome of a freezing pro- increased batch homogeneity. This result was expected because
cess is ice nucleation. Nucleation is a stochastic event, and the controlled nucleation results in the formation of larger ice crystals,
temperature at which nucleation occurs can vary widely when the and, thus, reduced risk of surface-induced denaturation. However,
traditional freezing techniques are applied, even within the same the controlled nucleation technique was not found to be equally
batch. As the nucleation temperature defines size, number, and advantageous during drying, and a satisfactory explanation for this
morphology of ice crystals, spontaneous nucleation leads to a high observation was not provided. In another study, 29 the application of
intervial variability, causing heterogeneous behavior during drying, controlled nucleation had neither a positive nor a negative impact
as well.12 on the physicochemical properties and long-term stability of 2 IgG1
Several methods have thus been proposed to control nucleation. antibodies. In the case of high antibody concentration, reduced
The earliest one was electrofreezing, 13 which uses a high-voltage particle formation was observed in samples frozen with a
pulse to generate an ice nucleus on a platinum electrode, that af- controlled nucleation approach, but the addition of a surfactant had
terward initiates ice crystallization. However, the need for indi- a much higher stabilizing effect. Therefore, it seems that controlled
vidual electrodes in each sample and, most importantly, the ice nucleation produced significant benefits on drying duration,
presence of an electrode in direct contact with the product is not product appearance, batch homogeneity, etc., whereas no dramatic
practical for good manufacturing practice product manufacture. improvements were observed for other applications (e.g., small and
14-18
Subsequently, the ultrasound technique was developed, which large molecules).
induces nucleation by an ultrasound transducer. However, this The objective of this work is, therefore, to provide further
technique, as the electrofreezing one, requires the installation of insight into the effects of controlled nucleation on protein stability
expensive additional equipment, and this hinders its applicability during the freeze-drying process. Moreover, a comparison will be
in manufacturing. A technique that overcomes this limitation is the made between trehalose and cellobiose as protein stabilizers, using
depressurization method, 19 which involves pressurization at about mannitol as bulking agent. Mannitol is among the most commonly
3 bar and subsequent depressurization inside the chamber to used bulking agents, and trehalose is often selected as lyopro-
induce nucleation of ice. This technique is applicable only for freeze tectant in protein formulations. Cellobiose is not equally common
driers that are designed to withstand high pressure because in pharmaceutical formulations, and was used in this work with the
equipment-adaptation is very cost-intensive. An alternative aim to evaluate its potential as a protein stabilizer. The high
method, named ice fog technique, was also proposed, 20,21 in which mannitol content should ensure quantitative crystallization during
a flow of cold nitrogen is released within the vacuum chamber, thus freezing, whereas the protein remains immersed in the amorphous
generating a suspension of small ice particles. Penetration of this sugar phase. This system is easy to be freeze-dried, and is also
ice fog into the vials induces ice nucleation. This approach is representative of the low-density, partially crystalline cakes that
currently available for use in commercial dryers manufactured by are typical of many pharmaceutical products; it represents there-
IMA Life,22 while specific optimization is required if the controlled fore a suitable model formulation. However, controlled nucleation
ice nucleation set is being retrofitted to an already existing dryer. may be extremely beneficial also for fully amorphous higher-
In this study, vacuum-induced nucleation (VIN), or vacuum- concentrated systems, which are more difficult to process. The
induced surface freezing (VISF), was used as previously described application of VISF to amorphous high-concentrated systems will
by Oddone et al. 23,24 According to this technique, during freezing, be the subject of future investigations.
the pressure inside the drying chamber is reduced to a low value For this study, we chose as model protein the human growth
(~1-2 mbar) for a short time, generally less than 1 min, until hormone (hGH), an aggregation-prone protein widely studied in
nucleation is induced in all vials. This reduction in pressure causes the literature. 30-36 The extent of protein aggregation after freeze-
partial evaporation of water and, thus, a fast reduction in product drying, carried out using either spontaneous or VIN, has been
temperature, that promotes the nucleation of ice. With respect to chosen as benchmark to investigate the impact of the selected
the original approach by Kramer et al., 25 Oddone et al. proposed the freezing protocol on protein stability. The impact of the nucleation
isolation of the drying chamber from the condenser, once the conditions on the bioactivity of the lyophilized hGH was also
desired value of vacuum is reached. This modification aims to assessed by measuring cell proliferation in the lactogenic
reduce the blow-out phenomena, by letting pressure increase lymphoblastic rat cell line, Nb2-11. 37 From the results obtained, we
within the freezing chamber during the vacuum period because of will demonstrate that the VISF technique can improve process
water evaporation. The result is that the elegance of the final performance, without having negative effects on protein aggrega-
product is remarkably improved, thus making VISF much more tion or bioactivity.
256 I. Oddone et al. / Journal of Pharmaceutical Sciences 109 (2020) 254-263

Table 1 maximum). These were detected in reversing, nonreversing, and

Details of the Freeze-Drying Cycles total heat flow curves with a difference between them of 0.2 C to
Number of Vials Freezing Tn , C 0.5 C.
Protocol For the dynamic mechanical analyses, a DMA 800 (TA In-
hGH-T þ M hGH-C þ M TþM CþM
struments) was used. 38 A small volume (200 mL, approximately 200
1 10 10 48 48 Spon. n. a.
mg) of the solution of interest was pipetted onto one surface of a
2 10 10 48 48 VISF 5
3 / 5 / 345 Spon. n. a. steel sample tray which was then clamped and inserted into the
4 / 5 / 345 VISF 5 DMA. Cooling of the system was provided by evaporation of liquid
5 / 5 / 345 VISF 10 nitrogen. The storage modulus and tan d values were recorded and
6 / 5 / 345 Spon./Anneal. n. a.
plotted against temperature (between 70 C and þ10 C), at 1 Hz
7 21 17 31 31 Spon. n. a.
8 21 17 31 31 VISF 5
oscillation frequency. The dynamic amplitude and scan rates were
50 mm and 1 C/min, respectively. The glass transition temperature
was determined based on peaks in the tan d values.
For the FDM analyses, a small amount (5 mL) of each formulation
Materials and Methods
was pipetted onto a special quartz crucible, using a metal shim and
coverslip. The crucible was then mounted on a Linkam FDCS196
Preparation of Solution
Cryostage and analyzed using an Olympus BX51 microscope with
plane polarized light. Images were captured by a charge-coupled
Details of the freeze-drying cycles performed in the present
device camcorder. This process was programmed and controlled
work are reported in Table 1.
(Linkam Scientific Ltd., Tadworth, Surrey, UK). Samples were frozen
For all the cycles, the stability of the hGH was evaluated in the
to 50 C at 5 C/min. Subsequently, pressure was lowered to 10
presence of either 6 mg/mL trehalose (T) or 6 mg/mL cellobiose (C).
Pa, and the sample was heated at 2 C/min until collapse was
In both cases, mannitol (M), at 42 mg/mL concentration, was used
observed. The presence of an annealing step at 10 C (for 30 min)
as bulking agent. The objective of this work was to compare 2
was also considered in 2 tests. The system was calibrated using 5%
freezing protocols (spontaneous and controlled nucleation), and 2
w/v trehalose in water, and single determinations were made for
different stabilizers (cellobiose and trehalose), focusing on protein
each formulation.
stability. The effect of the freezing protocol should mainly be
related to the ice crystal size, and therefore specific surface area, of
Preparation of Batches
the cake. The ice crystal size is affected by several factors, encom-
passing both the freezing protocol (cooling rate, nucleation tem-
Details on the number of vials within every batch are shown in
perature, presence of an annealing step), and the formulation. The
Table 1. In any case, care was taken to have the active (hGH-con-
addition of a bulking agent, such as mannitol, as dominant
taining) vials in the middle of the batch, and therefore shielded by
component (42 vs 6 mg/mL) makes it possible to rule out the effect
other vials, so as to avoid edge effects. This should guarantee a more
of having a different stabilizer (cellobiose vs. trehalose). Therefore,
homogeneous behavior of active vials in different batches and
the ice crystal size should be influenced solely by the freezing
during different cycles because of a similar exposure to external
protocol. This allows a clearer separation of the contributions of
radiations.
formulation and freezing protocol on protein stability.
Each vial (5 mL screw capped vial Schott part VC0005 internal
All the formulations were prepared in 1.1 mM sodium phos-
diameter 14 mm; Adelphi Healthcare Packaging, Haywards Heath,
phate buffer, pH 7.8. The hGH used was either the NIBSC standard
UK) was filled with 1 mL of solution. In all cases, the excipient
(WHO International Standard Somatropin, NIBSC code: 98/574) for
cycles 3-6, or a commercial time-expired recombinant product
formulations were filtered before filling using 0.2 mm filters,
whereas the hGH-containing solutions were not filtered, to avoid
supplied by NIBSC (for cycles 1-2 and 7-8), and was dialyzed for
loss of protein during the process. However, hGH was always added
about 24 h against the selected formulations using a dialysis kit
to a prefiltered formulation.
(Slide-A-Lyzer, 3.5 kDa cutoff; Thermo Fisher Scientific, Hemel
In each batch, the temperature profile inside 3 different vials,
Hempstead, UK). Dialysis was carried out under controlled tem-
containing the placebo formulations, was monitored by means of
perature conditions (4 C). At the end of the dialysis, the concen-
T-type copper and constantan miniature thermocouples. Thermo-
tration of hGH was determined by UV spectrophotometry, and the
couples were placed at the bottom center of the vial, and touching
dialyzate was diluted so as to obtain a concentration of hGH of
the bottom.
approximately 0.5 mg/mL (cycles 1-6 in Table 1) or 0.25 mg/mL
(cycles 7, 8 in Table 1).
Freeze-Drying Cycles

Characterization of the Formulations The freeze-drying cycles were carried out using either the Lyo-
Beta 15 (Telstar, Terrassa, Spain), for cycles 1-2, or 25 (Telstar), for
Modulated differential scanning calorimetry (MDSC), dynamic cycles 3-6, or the Virtis Genesis 25EL (Biopharma Process Systems,
mechanical analysis (DMA), and freeze-drying microscopy (FDM) Winchester, Hampshire, UK) freeze-drier, for cycles 7-8. The
analyses were performed on the 2 formulations under freezing protocols used in this work were the conventional shelf-
investigation. ramped freezing (spontaneous nucleation), and the VISF
MDSC was performed using a Q2000 calorimeter (TA In- (controlled nucleation). In one case (cycle 6 in Table 1), an
struments, Elstree, UK). Large volume (100 mL) stainless steel pans annealing step was performed after spontaneous nucleation. The
were used and 80 mL aliquots were sealed inside them and analyzed annealing step was performed at 10 C, with a duration of 30 min.
against an empty pan as reference. Samples were frozen to 90 C As regards spontaneous freezing, a 0.5 C/min ramp to 45 C wa s
at 10 C/min, and then heated to 25 C at 1.5 C/min, with modula- performed. The product was then kept at 45 C for 2 h. By contrast,
tion at 0.23 C/min. Results were analyzed using Universal Analysis for VISF, the batch was equilibrated at T n, as detailed in Table 1, for 1 h.
software (TA Instruments), and the critical thermal event temper- Pressure inside the chamber was then lowered until nucleation was
ature was assigned based on exotherms in the profiles (at peak observed (1 mbar). When all the vials nucleated (<1 min), pressure
 I. Oddone et al. / Journal of Pharmaceutical Sciences 109 (2020) 254-263 257

was quickly released to the atmospheric value. For cycles performed A-1 Envirosciences) and titrations deemed valid only if a 50 mL
in this work, it took less than 1 min to reach atmospheric pressure. It injection gave a water result of 180-210 mg with a relative standard
should be considered that pressure should be released as quickly as deviation less than 5% over 3 consecutive titrations. Three placebo-
possibleto avoid boiling of the solution. This result can be achievedby containing vials were analyzed for each formulation, and both the
quickly increasing the chamber pressure to a value above the vapor average value and relative standard deviation on the residual
pressure of water at T n. The subsequent rise to atmospheric value can moisture could be calculated.
occur more slowly, without any damage for the product. The product
was then kept at 10 C for 1 h. Finally, temperature was decreased SEM Analysis
to 45 C and held for 2 h.
The 1 h hold at 10 C for the VISF approach is different from an In the case of cycles 3-8, the pore dimension of the products
annealing step. In a common annealing step, an already frozen obtained after freeze-drying was analyzed using a scanning elec-
product is brought to higher temperature (above the glass transi- tron microscope (SEM, FEI type; Quanta Inspect 20 0, Eindhoven,
tion) to allow crystallization of formulation components and crystal the Netherlands). The samples were cut along their vertical axis,
growth because of Ostwald ripening. By contrast, in the case of the and 3 SEM images were taken at the top, center, and bottom of the
1 h hold at 10 C for VISF, the product has nucleated already, but is cake.
still not completely frozen. The objective, in this case, is to allow a Afterward, approximately 10 0 pores were selected in each im-
slow growth of the ice crystals, so as to obtain a large ice crystal age, and each of them was approximated to an ellipse. The diameter
size. This process occurs slowly, and enough time must be given for of the circle having the same area to perimeter ratio of the
the ice crystal growth to complete. approximating ellipse was then assumed as pore dimension, and
For all the cycles, primary drying was carried out at 35 C and 7 the numerical average (with a corresponding standard deviation) of
Pa (0.07 mbar) while during secondary drying temperature and the obtained distribution was assumed as the average pore size, D p,
pressure were 20 C and 7 Pa, respectively. A 10 h ramp from the of the product.
primary to secondary drying temperature was used, to reduce the
risk of collapse. X-Ray Diffractometry
During drying, the pressure inside the drying chamber was
monitored by means of both a capacitance (MKS Baratron, cycles 1- For cycles 7-8, X-ray diffractometry was also performed on the
6) and a thermal conductivity (Pirani, cycles 1-8) manometers. The placebo formulations, to identify the polymorphic state of mannitol
capacitance manometer always measures the correct value of in the dried samples. X-ray diffractometry (XRD) patterns were
pressure inside the chamber, whereas the Pirani gauge is calibrated obtained using a PANalytical X’Pert (Cu Ka radiation) diffractom-
in nitrogen and its readings are therefore shifted to higher values eter, within 5 -65 as 2q range. The data were acquired at each
during the primary drying phase, when water vapor is present in 0.026 . The resulting diffractograms were analyzed using the
the drying chamber as a result of sublimation. HighScorePlus program (vers. 3.0.5), by comparison with the X-ray
By compariso n of the 2 pressure p rofiles, it is also possible to diffraction patterns of the reference materials ( a , b , and d-anhy-
calculate the on set and offset ti mes. 39 The onset cor responds to drous mannitol).
the time at whic h the Pirani gauge rea ding begins to decay and
decline, and indicates that sublimation has ended in a not negli- HPLC-SEC Analysis
gible number of vials. By contrast, at the offset time, the ratio
between the cap acitance and Pi rani readings equals 1, meaning The soluble aggregates of hGH were measured by size exclusion
that sublima tion has ended i n all the vials. T he offset mark s the chromatography (SEC). High-performance liquid size exclusion
end of the prim ary drying phase, where as the difference b etween chromatography (HPLC-SEC) analysis was performed on dried
offset time an d onset time (in the fo llowing referred to as o nset- samples, for all the freeze-drying cycles performed. The freeze-
offset time) is an i ndicator of within- batch heterogeneit y. The dried samples were reconstituted to 1 mL using ultrapure water
larger it is, the more th e sublimation beh avior of vials within the and then centrifuged for 5 min at 13,000 rpm. A TSKgel G3000
batch is different. SWXL column (Tosoh Bioscience, Reading, UK) was used on a
The ramp to secondary drying was started after the offset Thermo Dionex U3000 HPLC system (Thermo Fisher Scientific)
time, which can be considered as the end of the sublimation using a flow rate of 0.6 mL/min, a run time of 50 min and UV
process. By contrast, the duration of secondary drying was, in all detection at 214 and 280 nm. The mobile phase used was 2-
cases, set to 5 h. propanol R, 0.063 M phosphate buffer solution pH 7.0 R (3:97 V/
V), as suggested in European Pharmacopoeia 9.0. 40 For all batches, 3
Residual Moisture Analysis hGH-T þ M vials, when present, and 3 hGH-C þ M vials, were
analyzed. For cycles 1-3 and 5-6, 1 hGH-T þ M vial, when present,
In the case of cycles 7-8, the residual moisture of dried samples and 1 hGH-C þM vial were analyzed dividing the top part of the
was evaluated using automated Karl Fischer titration. Sample am- cake from the bottom. This was carried out to quantify the degree of
poules were opened in a low relative humidity environment using a intravial heterogeneity in aggregate formation.
glove bag (Captair Pyramid, Cole-Parmer, London, UK) flushed with
dry nitrogen to an RH level of 5%-10%. Cell Culture
Coulometric Karl Fischer titrations were performed using an
automated Karl Fischer system, based on the AquaFast system (A1- Rat Nb2-11 cells (rat lymphoblast cell line, European Collection
Envirosciences, Blyth, UK). Samples of lyophilized material were of Authenticated Cell Cultures, Public Health England, Salisbury,
dispensed into septum-sealed autosampler vials in the glove bag UK) were routinely grown in suspension culture in Fischer’s me-
environment and then reconstituted remotely with a known dium containing 10% (v/v) heat inactivated fetal bovine serum, 10%
aliquot of anolyte. After a period of time to allow insoluble material (v/v) horse serum, 0.075% (v/v) sodium bicarbonate, 50 mM 2-
to settle, an aliquot of the supernatant was titrated into the mercaptoethanol, 50 units/mL penicillin, and 0.05 mg/mL strepto-
coulometric Karl Fischer cell. The coulometer was calibrated using mycin. Fischer’s medium, heat inactivated horse serum and 2-
the Aquamicron P water standard solution (4 mg/g water content, mercaptoethanol were obtained from Thermo Fisher Scientific.
258 I. Oddone et al. / Journal of Pharmaceutical Sciences 109 (2020) 254-263

Fetal bovine serum was obtained from BioSera Europe (Nuaille, Results
France) and sodium bicarbonate (7.5%), penicillin, and streptomycin
from Sigma Chemical Co., Ltd. (Poole, UK). Cells were maintained by Thermal Characterization of the Formulations
passaging every 4-5 days at a cell density of approximately 1 10 4
’
cells/mL and incubated at 37 C in an atmosphere of 5% CO 2 . At 16- In Table 2, the glass-transition temperature T g (mid-point) as
20 h before assay, cells were prepared in culture medium without measured by MDSC and DMA, and the collapse temperature T c as
penicillin and streptomycin at a cell density of 2 10 5 cells/mL and measured by FDM are shown. In absence of annealing and with a fast
incubated as aforementioned. cooling and heating rate, the formulation with the lowest collapse
temperature was the cellobiose-based one, which showed a first
microcollapse at about 35 C, and complete collapse around 29 C.
Bioassay The collapse temperature observed for the trehalose-based
formulation was about 3 C higher ( 26.5 C). Results obtained by
The assay is based on the quantitation of ATP in metabolically freeze-drying microscopy are correlated with the glass transition
active cells. The International Standard (WHO International Stan- temperature, as measured by both MDSC and DMA. These 2 tech-
dard Somatropin, NIBSC code: 98/574) and test samples containing niques showed good accordance and revealed a higher critical
0.25 mg hGH lyophilized using spontaneous or vacuum-induced temperature in the case of trehalose as excipient.
nucleation (cycles 7 and 8 in Table 1) with a cellobiose and If an annealing step is introduced (T þ M, annealing and C þ M,
mannitol excipient (C þ M SPON and C þ M VIN) or trehalose and annealing in Table 2), or if a slow cooling and heating rate is used,
mannitol excipient (T þ M SPON and T þ M VIN) were recon- the crystallization of mannitol is promoted. In this case, the
stituted, aliquoted in low-protein binding microfuge tubes (Cata- formulation is stable also at higher temperatures. The FDM allows
logue number 0030108094, Eppendorf Ltd., Stevenage, UK) and identification of the melting temperature of the crystalline phase,
stored at 30 C. Dilutions of the WHO International Standard for which was found to be around 5 C in presence of trehalose,
Somatropin, 98/574, and test samples were prepared in assay me- and 7 C in the case of cellobiose as amorphous stabilizer.
dium that comprised Fischer’s medium containing 1% (v/v) horse If the objective were to optimize the cycle duration, primary
serum, 0.075% (v/v) sodium bicarbonate, and 50 mM 2- drying should be performed at the maximum allowable product
mercaptoethanol. Nb2-11 cells, pretreated as described previ- temperature which, as illustrated, is a characteristic of the product,
ously, were washed twice with phosphate buffered saline (pH 7.2), and also depends on the presence of an annealing step for crys-
suspended in assay medium at a density of 1 10 5 cells/mL and talline components. A different drying temperature would, how-
transferred (50 mL/well) to a 96-well microtitre plate (Greiner Bio- ever, result in different thermal stresses for the protein. Therefore,
One, Stonehouse, UK, code 655180). Dilutions of standard and test as the focus here was on the effect of the freezing protocol on
samples were added as an additional 50 mL volume to each well. protein stability, primary drying was carried out at the same tem-
The maximal cell response was assessed by stimulating with a 10 perature ( 35 C) for all the formulations and freezing protocols
ng/mL concentration of the International Standard, 98/574. Hor- under investigation.
mone concentrations are expressed as the final concentration in the
wells.
The cells were in cubated for 30 ± 2 h in conditions described Performance of the Freezing Protocols
previously. Proli feration was then estimate d by the add ition of a
100 mL volume of Cel lTitre-Glo reagen t to each well and in cu- Previous studies have shown that the use of controlled nucle-
bating with s haking in the da rk at room temperature . CellTiter- ation, and in particular of the VISF, resulted in a marked reduction
Glo ® Luminescent Cell Viability Assay reagent was obtained from of primary drying time. 23,41 This was confirmed by the results ob-
Promega UK (Sou thampton, UK). Th e resulting cell extr acts were tained in this work. As shown in Table 3, the drying time (difference
then transferre d to a white 96-well plate (Grein er Bio-One code between the offset of the Pirani decay curve and the initiation of
655075), incubated at room temperature in the dark for 15 min drying) for cycles performed using controlled nucleation was 10%-
and the lumine scence measure d using a SpectraM ax M5 micro- 40% shorter than in the case of conventional freezing. This is due to
plate reader (M olecular Devic es (UK) Ltd., Wokingha m, UK) the higher nucleation temperature in the case of VISF, which leads
controlled by sof tware SoftMax P ro 6 (Version 6.4, 1992-2014 to the formation of larger pores. In turn, big pores facilitate water
Molecular Devices LLC). Eac h test dil ution was measu red in trip - vapor diffusion through the dried layer and allow a higher subli-
licate and test s amples were asses sed in 2 indepen dent bioassays. mation rate. This is also confirmed by the SEM analysis, Table 4 and
Relative bioactiv ity was assessed by fitting a sigmoidal curve using Figure 1, which evidenced the presence of larger pores in samples
CombiStats, version 5.0 (1999-2013 EDQM and Council of Eu rope, obtained by controlled nucleation. The introduction of an annealing
Strasbourg, France) . step after spontaneous nucleation, as in cycle 6, increased the pore
size and reduced the drying time approximately to the same extent
as VISF. This is due to the Ostwald ripening phenomena, which
Table 2
Results of MDSC, DMA, and FDM Performed on the 2 Formulations Under
Investigation
Table 3
0
Tg, C (Mid-point) T ,c C Drying Time (Difference Between the Offset of the Pirani Decay Curve and the
Initiation of Drying) and Onset-Offset Times (Difference Between Offset Time and
MDSC DMA FDM Onset Time) for the Freeze-Drying Cycles Performed
TþM 27 26 26.5 (collapse)
Freezing Protocol Drying Time, h Onset-Offset Time, h
CþM 28 28 35 (microcollapse),
29 (collapse) 1 Spon. 42 12
T þ M, annealing 5 2 VISF 27 7
C þ M, annealing 7 3 Spon. 47 13
4 VISF 35 5
For the last 2 tests, an annealing step at 10 C was included, and the collapse
5 VISF 38 4
temperature was measured by FDM.
6 Spon./Anneal. 35 6
MDSC, modulated differential scanning calorimetry.
Citations (11) References (56)

... Moreover, even though economic evaluations have been presented in the past [9][10][11], they are often too broad or
too specific to be used for making comparisons between different process strategies. Whereas little effort has been
made in this field to determine the economic impact of freeze-drying quantitatively, the optimization of the process has
usually remained product-or quality-specific [12][13][14] [15] [16][17][18], with no considerations of possible bottlenecks
in the expenses. In this work, we aim to create a model to evaluate the actual economic impact of a freeze-drying cycle,
and evaluate the impact of process parameters on processing costs and, finally, the benefits of process optimization. ...
... At the end of the freezing step, the product and the components of the drying chamber are at temperature T F ,
usually below −50 • C. During primary drying, the heat required to increase their temperature and allow ice sublimation
reads: Q PD,load = Q PD,prod +Q PD,vials +Q PD,shelf +Q PD,oil (15) which is similar to Equation (2) except the
terms correlated to the air and the chamber walls are missing. The air will not be considered because,during primary
drying, the freeze-drier works under vacuum, while the temperature of the chamber walls is controlled by the heat
coming from outside the freeze-drier and, thus, no heat has to be supplied directly. ...

Economic Analysis of a Freeze-Drying Cycle

Article Full-text available
Nov 2020
Lorenzo Stratta · Luigi Carlo Capozzi · Simone Franzino · Roberto Pisano

View Show abstract

... The possibility to control the nucleation temperature during freeze drying would therefore make it possible 61 to
modulate the overall process efficiency, and the critical attributes of the final product as well. Moreover, 62 the batch
homogeneity, which is a key requirement in the pharmaceutical and reference standard industries, (Oddone et al.,
2020) . In this study we will look at the impact of induced nucleation 68 on the reconstitution time of human plasma after
freeze drying and the recovery of Factor VIII activity in that 69 plasma. ...
... 334between the controlled and spontaneously nucleated samples was detected. These results are in line with 335
our previous study (Oddone et al., 2020) , where HPLC-SEC and a cell-based potency assay seemed to give 336
evidence for no dramatic difference in the behaviour of hGH at low concentration when either VISF or 337 spontaneous
nucleation were used. Our results for the highly concentrated plasma system, combined with338 previously published
data reporting a negligible effect of controlled nucleation on protein stability in low 339 concentrated systems (Oddone
et al., 2020; Vollrath et al., 2018), seem to suggest that the benefits of 340 controlled nucleation may depend on
concentration. ...

Impact of Controlled Vacuum Induced Surface Freezing on the Freeze Drying of Human Plasma
Article
Mar 2020 · INT J PHARMACEUT
Andrea Arsiccio · Paul Matejtschuk · Ernest Ezeajughi · Roberto Pisano

View Show abstract

... In addition to influencing process performance, freezing is also known to impact the stability of incorporated
biomolecules and protein activity (Arsiccio, Giorsello, et al. 2020; Oddone et al. 2020; Fang et al. 2020;Susa et al. 2021).
This is true for monoclonal antibodies which are prone to stress during freezing due to phase separations and the
formation of multiple interfaces between the biomolecule and ice crystals leading to physical or chemical instabilities. ...
Recent advances and persistent challenges in the design of freeze-drying process for monoclonal antibodies
Article
Oct 2022
Hassana Hsein · Julie Auffray · Thierry Noel · Pierre Tchoreloff

View Show abstract

... Among such techniques, vacuum-induced surface freezing (VISF) has been shown to improve process efficiency and
product homogeneity [10][11][12][13][14][15][16], and in some cases, to have either a negligible [17] or a positive [18]
effect on protein recovery. For the application of VISF, the product is first equilibrated at a temperature T n above the
onset of spontaneous nucleation, then the chamber pressure is subsequently reduced and held for a short amount of
time t n (<1 min) to a product-specific value P n (generally, around 1 mbar [10]). ...

Surface Treatment of Glass Vials for Lyophilization: Implications for Vacuum-Induced Surface Freezing
Article Full-text available
Oct 2021
Francesco Regis · Andrea Arsiccio · Erwan Bourles · Roberto Pisano

View Show abstract

... The freezing step has been recently studied in detail as it was proven to substantially influence the product structure
[4][5][6] and the drug residual activity [7][8] [9] [10]. When water is cooled below its equilibrium temperature, it can
remain in the liquid state for a relatively long time before the occurrence of the phase transition into solid ice. ...

Investigation of the Freezing Phenomenon in Vials Using an Infrared Camera

Article Full-text available
Oct 2021
Maite Harguindeguy · Lorenzo Stratta · Davide Fissore · Roberto Pisano

View Show abstract

... Although solid-state properties differed slightly, they concluded that controlled ice nucleation had neither a positive
nor negative impact on protein stability compared to stochastically nucleated samples. Oddone et al. reported no
difference in stability of stochastic versus controlled nucleation vials by the "partial vacuum" method investigated for
different formulations of human growth hormone [22] . The current study focused on investigating and comparing the
impact of three mechanistically different nucleation techniques on protein stability and included challenging protein
configurations. ...

Comparison of Techniques to Control Ice Nucleation during Lyophilization

Article Full-text available
Nov 2020
Jacob Luoma · Erika Ingham · Carmen Lema Martinez · Andrea Allmendinger

View Show abstract

... [21] Smaller ice crystals with larger interface would lead to a higher risk of activity loss. Oddone et al. [22] found that
larger ice crystals obtained by vacuum induced surface freezing are beneficial for the bioactivity of human growth
hormone. ...

Quantitative investigation on the effects of ice crystal size on freeze-drying: The primary drying step
Article Full-text available
Aug 2020
Luo Chun · Zhiqiang Liu · Sha Mi · Longquan Li

View Show abstract

... The use of controlled (or induced) ice nucleation in freeze-drying is receiving increased attention due to the
advantages it can offer in terms of increasing sublimation efficiency through control of the ice nucleation temperature,
which affects product porosity and uniformity [12,13] and may also reduce activity losses in cases where ice formation
or freeze-concentration effects are known to induce protein denaturation [14][15] [16] . Any method that allows for a
control of the nucleation temperature results in several degrees of freedom for the freeze-drying process. ...

The Principles of Freeze-Drying and Application of Analytical Technologies

Chapter
Jan 2021
Kevin Ward · Paul Matejtschuk

View Show abstract

 ... 213 The benefits of vacuum induced surface freezing (VISF) on the stability of the human growth hormone (hGH)
were also investigated. 214 In this case, both HPLC-SEC and a cell-based potency assay indicated that there was no
dramatic difference in the behaviour of hGH at low concentration when either VISF or spontaneous nucleation were
used. In the case of very unstable molecules, such as myoglobin at low pH, the application of VISF proved to be
detrimental. ...

The Ice-Water Interface and Protein Stability: A Review

Article
Mar 2020
Andrea Arsiccio · Roberto Pisano

View Show abstract

Development of freeze-drying cycle via design space approach: a case study on vaccines
Article
Aug 2020
Bernadette Scutella · Erwan Bourles

View Show abstract

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