Hydroxypropyl beta cyclodextrin: a water-replacement agent or a surfactant upon spray freeze-drying of IgG with enhanced stability and aerosolization

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Hydroxypropyl beta cyclodextrin: a water- replacement agent or a surfactant upon spray freeze-drying of IgG with enhanced stability and aerosolization

Shahriar Milani, Homa Faghihi, Abdolhosein Roulholamini Najafabadi,

Mohsen Amini, Hamed Montazeri & Alireza Vatanara To cite this article: Shahriar Milani, Homa Faghihi, Abdolhosein Roulholamini Najafabadi, Mohsen

Amini, Hamed Montazeri & Alireza Vatanara (2020): Hydroxypropyl beta cyclodextrin: a water- replacement agent or a surfactant upon spray freeze-drying of IgG with enhanced stability and aerosolization, Drug Development and Industrial Pharmacy, DOI: 10.1080/03639045.2020.1724131

To link to this article: https://doi.org/10.1080/03639045.2020.1724131

Published online: 17 Feb 2020.

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Hydroxypropyl beta cyclodextrin: a water-replacement agent or a surfactant upon spray freeze-drying of IgG with enhanced stability and aerosolization

Shahriar Milania, Homa Faghihib, Abdolhosein Roulholamini Najafabadia, Mohsen Aminic, Hamed Montazerib and

Alireza Vatanaraa aDepartment of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; bSchool of Pharmacy-International

Campus, Iran University of Medical Sciences, Tehran, Iran; cDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of

Medical Sciences, Tehran, Iran ABSTRACT The great potential of hydroxypropyl beta-cyclodextrin (HPßCD), as a dried-protein stabilizer, has been attributed to various mechanisms namely water-replacement, vitrification and surfactant-like effects.

Highlighting the best result in our previous study (weight ratio IgG: HPßCD of 1:0.4), herein we designed to evaluate the efficacy of upper (1:2) and lower (1:0.05) ratios of HPßCD in stabilization and aerosol prop- erties of spray freeze-dried IgG. The protective effect of HPbCD, as measured by size exclusion chromatog- raphy (SEC-HPLC) was most pronounced at C30 and C300, IgG:trehalose:HPbCD ratios of 1:2:0.25 and

1:2:0.05 with aggregation rate constants of 0.46 ± 0.02 and 0.58 ± 0.01 (1/month), respectively. The second- ary conformations were analyzed through Fourier transform infrared spectroscopy (FTIR) and all powders well-preserved with the lack of any visible fragments qualified through sodium dodecyl sulfate polyacryl- amide gel electrophoresis (SDS-PPAGE). Scanning electron microscopy (SEM) and twin stage impinger (TSI) were employed to characterize the suitability of particles for further inhalation therapy of antibodies and the highest values of fine particle fraction (FPF) were achieved by C30 and C300, 56.43 and 48.12%. The powders produced at the current ratio 1:2:0.25 and 1:2:0.05 are superior to our previous examination with regards to manifesting lower aggregation and comparable FPF values.

ARTICLE HISTORY Received 15 August 2019 Revised 17 January 2020

Accepted 25 January 2020 KEYWORDS Spray freeze-drying; IgG;

HPbCD; surface activity; direct binding; aerosolization

Introduction Spray freeze drying (SFD) has been emerged as a drying technol- ogy to prepare stable solid dosage forms of pharmaceutical prod- ucts with unique particle properties (e.g. suitable for aerosol delivery). Specifically, SFD was shown to produce desirable, fine, uniform and porous powders of proteins without affecting the pri- mary characteristics [1]. Additionally, in the case of heat-sensitive proteins, SFD can be a fine alternative to other drying techniques such as spray drying.

Although SFD is regarded as an effective method to preserve protein stability, excipient-free formulations of protein powders were prone to different types of instabilities [2]. Atomization from the nozzle of spray, sensitivity to air–liquid interfaces, cold- denaturation, phase separation and ice crystallization are stresses which might be inserted on proteins via spraying and freeze-dry- ing steps respectively [3,4]. Dehydration is another important chal- lenge that protein encounters and may lead to conformation disturbance and protein intermolecular aggregation. Suitable exci- pients should be therefore added to mitigate the stresses exerted on IgG molecules. Also, in the case of processing of powders for respiratory delivery, the ability of additives to augment the aero- solization efficiency is crucial.

As a group of well-known stabilizing agents, sugars such as tre- halose and sucrose (disaccharides) are employed acting through preferential exclusion mechanism within the dehydration process [1]. Additionally, they can rise glass transition temperature (Tg) of proteins and well-preserve them from various disturbances at long-term storage. Also, CDs (cyclic oligosaccharides) are capable of forming stable complexes with aggregation-prone hydrophobic moieties of proteins, taking part in hydrogen bonding with pro- teins and representing intrinsic surfactant-like effect [5]. They have also been reported as enhancers for fine particle fraction (FPF) in some inhalable formulations like salbutamol and budesonide [6,7].

Among various derivatives of CDs, the superiority of HPbCD has been clearly established and is believed to be mostly attributed to the polar 2-hydroxypropyl groups in CDs backbone. HPbCD has been reported to actually demonstrate surface-active properties that are required for effective surface protection of proteins via

SFD [8–10].

Analyzing the impact of HPbCD on protein aggregation, it can be inferred that depending on the investigated protein, manufac- turing process and the nature of related stress conditions, the obtained result can be varied. CDs have been accordingly used in a broad range varying from 0.0001% up to 10% w/v implying the fact that they can fulfill different roles based on their quantity [11,12]. To act as lyoprotectant, bind to protein and take part in water-replacement mechanism, CDs should be added at higher weight ratios from 1:1 to 1:5 of protein:sugar while their surfac- tant-effect needs lower amounts, less than 1% w/v [13].

A combination between sugars (trehalose and mannitol) with

CDs (HPbCD and bCD) was studied in our previous research to process stable IgG formulation, by SFD technique for future respiratory delivery [14].

In the presence of HPbCD, trehalose CONTACT Alireza Vatanara vatanara@sina.tums.ac.ir

Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran;

Homa Faghihi faghihi.h@iums.ac.ir School of Pharmacy-International Campus, Iran University of Medical Sciences, Tehran, Iran.

2020 Informa UK Limited, trading as Taylor & Francis Group

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY https://doi.org/10.1080/03639045.2020.1724131 could synergistically stabilize IgG powders incomparable to other preparations at the weight ratio IgG:trehalose:HPbCD of 1:1:0.4.

Although it was inferred that spray-freeze dried IgG was well- protected against aggregation in the mentioned ratio, rationally it would be of interest to explore the effect of HPbCD at lower and upper ratios as well. The complementary aim of the current design is to further interpret the dominant mechanism of action proposed to IgG molecules by CDs (whether the surfactant-effect or the sugar property).

To this end, we examined the weight ratio of IgG:HPbCD at three levels of 1:2, 1:0.25 and 1:0.05 to understand the underlying event through which CDs protect antibody via SFD. Secondly, dif- ferent ratios of IgG:trehalose (1:0.5, 1:1 and 1:2), more comprehen- sive than recent publication with a fixed ratio of 1:1, were employed to minimize the rate constant of IgG aggregation fol- lowing storage and concurrently optimize the aerodynamic behav- ior of the antibody powder.

Materials and methods Method Human IgG (150 KDa) was purchased from Interatect (Germany);

HPbCD was obtained from Acros (Belgium). Trehalose dehydrate, disodium hydrogen phosphate and sodium sulfate were pur- chased from Merck (Darmstadt, Germany) and all the other chemi- cals were obtained from

Sigma (USA) and were of analytical grades.

Preparation of SFD processed IgG samples Initially, human IgG was dialyzed against deionized water (cut off:

15 kDa) at 4 C overnight to achieve a pure IgG solution. Aqueous antibody solutions containing 200 mg IgG with different ratios of

HPbCD and trehalose were prepared (Table 1). To prepare a cryo- genic vapor for each experiment, a 2 L glass container was selected and filled with 0.4 L liquid nitrogen. Utilizing a lab-scale spray drier equipped with a

2-fluid nozzle (Buchi 191, Switzerland), the feed solution was atomized above the liquid nitrogen at 6 mL/min flow rate. Then, the formed slurry was placed on the bench until the remaining nitrogen was completely evaporated. The prepared frozen droplets were collected and lyophilized using a freeze drier (Christ, Germany). Primary drying took place at –50 C and 0.005 mbar for 24 h followed by a sec- ondary drying by gradually elevating the temperature to –20 C over a period of 24 h. The yield of powder recovery was estimated to be about 85%.

Size exclusion chromatography (SEC-HPLC) The content of soluble aggregate and remaining IgG monomer were evaluated by SEC-HPLC after the process and following stor- age periods. In brief, 20 mL of each formulation with IgG concen- tration of 2.5 mg/mL was filtered and injected to HPLC system equipped with a TSK 3000 SWXL column (Silica SEC phases with pore size of 5 lm, 7.8 mm  30 cm, Tosoh Biosep, Germany) and a pump (Jasco, USA). The absorbance was recorded using an UV detector at 280 nm. The set flow rate was 0.5 mL/min and the mobile phase consisted of a mixture of 0.1 M sodium sulfate and

0.1 M disodium hydrogen phosphate adjusted to pH: 6.8. The test was done in triplicate for each sample. Applying a suitable resin in a pre-packed column that can separate the monomer both from lower and higher molecular weight species was done in our studies. The peaks, A280 nm corresponding to aggregates and to monomer is quantified by integrating the peak areas and the per- cent of aggregates which was reported as follows [15]:

Soluble aggregates % ð Þ ¼ AUC aggregates % ð Þ AUC total peaks

% ð Þ (1) Kinetic of aggregation following sample storage at 45 C

To further assess the stability of freeze-dried antibody formula- tions, the amount of induced aggregation was calculated follow- ing 1 and 2 months of storage at 45 C and 60% of relative humidity. The rate of aggregation was determined by assuming both zero (linear regression of % aggregation versus time) and first-order (linear regression of log % aggregation versus time) kin- etics. The calculated R2 were compared for each sample. Since the corresponding values of R2 were higher considering first-order kin- etic of aggregation, the below equation was employed to measure the rate constant of aggregation:

Log soluble aggregates % ð Þ ¼ K time h ð Þ 2:303 6 logsoluble aggregates at time zero

% ð Þ (2) Fourier transform infrared spectroscopy (FTIR)

FTIR Spectra of formulations both after SFD and 2 months of stor- age were recorded using a Nicolet Magna Spectrometer (Thermo

Scientific, Waltham, MA, USA) at room temperature.

Approximately, 2 mg of formulations were thoroughly blended with 200 mg KBr and pelleted with a hydraulic press applying a force equal to 6–7 T. Results were then analyzed using Jasco

Spectra Manager software (Jasco Corporation, Oklahoma City, OK).

Amide I region (1700–1600 cm1) of spectra was analyzed for

Table 1. Composition of formulation components and the results of characterization.

No IgG (mg) Trehalose (mg) HPbCD (mg) b-sheet SFD (%) b-sheet

Storage (%) Aggregation 0 (%) Aggregation 1 mo (%)

Aggregation 2 mo (%) Rate constant aggregation (1/mo)

C1 200 100 400 77.82 78.15 0.10 ± 0.02 2.13 ± 0.12

14.77 ± 0.14 2.49 ± 0.15 C2 200 200 400 69.19 68.45

0.04 ± 0.01 0.60 ± 0.02 2.69 ± 0.23 2.10 ± 0.26 C3

200 400 400 72.59 72.34 0.01 ± 0.00 0.10 ± 0.02 0.69 ± 0.02

2.12 ± 0.39 C10 200 100 50 67.22 68.91 0.07 ± 0.01

2.06 ± 0.10 12.12 ± 0.23 2.58 ± 0.25 C20 200 200 50

65.13 66.73 0.10 ± 0.01 0.60 ± 0.01 2.17 ± 0.29 1.54 ± 0.12

C30 200 400 50 66.03 66.32 0.10 ± 0.01 0.15 ± 0.02

0.25 ± 0.05 0.46 ± 0.02 C100 200 100 10 66.99 66.40

0.10 ± 0.03 1.15 ± 0.02 11.38 ± 0.34 2.37 ± 0.16 C200

200 200 10 70.50 69.81 0.10 ± 0.01 0.21 ± 0.01 0.70 ± 0.02

0.98 ± 0.02 C300 200 400 10 66.39 67.22 0.09 ± 0.03

0.17 ± 0.01 0.28 ± 0.02 0.58 ± 0.01 The mean values of aggregation (%) as well as rate constants of aggregation have been reported.

2 S. MILANI ET AL. evaluation of the secondary structure of freeze-dried powders within the following steps: First, the scale range of wave number was adjusted in 1700–1600 (cm1). Then, the second derivation of deconvoluted spectra of ‘amide I’ band was prepared. Peak pro- cess was used to determine the height and width of each special peak.

Curve-fitting procedure was employed using a mixed

Gaussian/Lorentzian function. Then, the characteristics of each peak in definite regions were determined including peak width, height, centre, area and calculated percent of area. Finally, the total areas related to beta-sheet domain were added together to provide the final percent of beta-sheet structure in each formulation.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

The fragmentation and/or aggregation of the optimum formula- tion of IgG were evaluated immediately after preparation and also after 1 month and 2 months storage using SDS-PAGE, with poly- acrylamide gel 10%, according to Laemmli’s discontinuous method [16]. About 100 mg/mL diluted samples were mixed under non- reducing conditions with equal volumes of sample buffer and sub- sequently loaded on the gel. The gels were then run for 3 h at

100 V. Finally, the bands were stained by 0.1% Coomassie blue solution. Molecular weight standard markers included 11, 17, 25,

35, 48, 63, 75, 100 and 145 kDa.

Scanning electron microscopy (SEM) Morphological characteristics of processed IgG formulations were examined utilizing SEM (XL30, the Netherlands). Spray-freeze dried particles were first sputter-coated with gold (BAL-TEC, Switzerland) at room temperature and images were then taken at an accelerat- ing voltage of 30 kV. All samples were directly prepared for SEM after SFD without further processing.

Particle size and size-distributions of powders The aerodynamic diameters of spray-freeze dried powders were characterized by Malvern Mastersizer (UK) through suspending

10 mg of each formulation into 5 mL of acetonitrile as a solvent.

The mixture was then sonicated for 3 min in a water-bath sonica- tor (Starsonic, Italy). The average particle sizes were measured at obscuration from 0.15 to 0.20. The examination was performed 3 times for each powder. The Span, as the most common parameter to express the width of size distribution, was also reported through the below formula:

Span ¼ DV0:9DV0:1 DV0:5 (3) In-Vitro aerosol performance

In order to evaluate the aerosol performance of IgG powders after

SFD, a TSI (Copley Scientific, UK) was applied to estimate the emit- ted dose, ED and FPF of best-stabilized samples. Approximately,

10 mg of each sample was filled in size 2 HPMC capsules and placed in a CyclohalerV

R. The mouthpiece of TSI was linked with a vacuum pump (Copley Scientific, UK) in order to supply a flow rate of approximately 60 L/min for 5 s. Subsequently, the powder deposited in each stage was collected and measured using UV spectroscopy at 280 nm. ED was defined as the percent of loaded powder in capsules which was emitted by aerosolization (Equation (4)) and Fine Particle Dose (FPD) was determined as the percent of deposited particles in stage 2 of TSI (effective cutoff diameter < 6.4 mm). Finally, recovery dose was specified as the sum of particles regained from an inhaler device, capsule shells and accumulated particles in stage 1 and stage 2 of TSI. FPF is expressed as the ratio of Fine Particle Dose (FPD) to Emitted Dose (ED) and is expressed as percentage (Equation (5)) [17].

ED ¼ Recovered Dose ðRDÞFinal mass remaining in capsule

RD  100 (4) FPF ¼ FPD ED  100 (5) Stability evaluation following 1 month and 2 months storage at 45 C

SFD powders of IgG were placed in glass bottles and vacuum- sealed under 45 C and 60% relative humidity up to 2 months.

Subsequently, each sample was reconstituted in water for further evaluation of aggregate contents at appropriate time periods. The rate constant of aggregation for each formulation was calculated accordingly.

Results To investigate the most effective concentration of HPbCD on sta- bilizing IgG against aggregation along with promoting its aerosoli- zation properties, the combination of trehalose was examined with different ratios of HPbCD. Spray-freeze dried formulations were prepared under identical process parameters and then eval- uated with respect to aggregation, conformational stability and particle characteristics.

Physicochemical and structural stability of SFD processed

IgG powders SEC-HPLC after process and upon 1 as well as 2 months of storage at 45 C

The induced soluble aggregates were measured by SEC-HPLC method and the data are shown in Table 1. It was demonstrated that after SFD, the level of aggregation was in the range of

0.01–0.1% and the highest values of trehalose and HPbCD in C3, had a marked impact on the stabilization of IgG powder, inducing the lowest amounts of soluble aggregates (0.01%). From 0.10 to

2.13% of monomer loss was detected after 30 days of storage at

45 C and the highest one related to C1 with minimum and max- imum ratio of trehalose and HPbCD, respectively. The most mono- mer recovery was afforded by C3, C30 and C300 with aggregation values of 0.10 ± 0.02, 0.15 ± 0.02 and 0.17 ± 0.01%. The outcomes of the SEC-HPLC after 2 months of storage revealed high values of aggregates in C1, C10 and C100 up to even 14.77 ± 0.14%. The best

IgG protection provided by C30 and C300 with 0.25 ± 0.05 and

0.28 ± 0.02% of aggregation.

Rate constant of aggregation To assume the rate constant of aggregation, both zero and first- order kinetics were considered. For all preparations, the first-order one was suggested with regards to higher values of R2 for the

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 3 calculated equation of log aggregation (%) versus time (month).

The rate of aggregation varied from 0.46 ± 0.02 to 2.58 ± 0.25 (1/ month), Table 1. Concomitant with inferred data from 2 months of storage, the least aggregation rate constants resulted from C30 and C300 with constants of 0.46 ± 0.02 and 0.58 ± 0.01 (1/month), respectively. The highest speed of aggregate formation belonged to C1, C10 and C100 with rate amounts of 2.49 ± 0.15, 2.58 ± 0.25 and

2.37 ± 0.16, 1/month. The 2-month stored spectra of C10, C30 and

C300 were shown in Figure 1(A,B,C).

Secondary conformation of spray-freeze dried IgG powders

The basic secondary conformational features of IgG powders were examined through FTIR spectroscopy. The main type of secondary structure of IgG is dominantly composed of beta-sheet structures.

Bands in the region of amide I (basically 1640–1695 cm1) are contributed to the presence of beta-sheet [18]. The calculated percentage of beta-sheet in prepared powders was provided in

Table 1, which is in the range of 65.13–77.82% after SFD and

66.32–78.15% after storage condition.

The FTIR spectra of two selected formulations namely C10 and C30 were prepared in

Figure 2 (A,B), as formulations with respective highest and lowest rate of aggregation.

Chemical stability of processed IgG powders Considering fragmentation as one of the chemical degradation pathways of IgG molecules, SDS-PAGE analysis was designed to qualitatively characterize formulations.

The stained-gel was depicted in Figure 3. It could be inferred that after 2 months of sample incubation at 45 C the unique band at approximately

150 KDa was recognizable in all samples. There are no detectable bands at lower region indicating visible fragments in spray-freeze dried samples not only after process but also upon stor- age situation.

Characterization of formulations based on particle features

Surface morphology and particle size of spray-freeze dried IgG preparations

Since the particle flowability and friability is shown to be in direct relation with the morphology, the powders were characterized through SEM. In overall consideration, all samples presented a non-irregular structure with a significant degree of sphericity,

Figure 4. A common feature of powders was indicative of the per- meable highly-porous shape; however, the particles appeared to be different in terms of the level of porosity as well as average particle sizes. It can also be estimated that particle sizes were around and/or less than 10 mm. It seems that C1, as the formula- tion with the least amount of trehalose and the highest ratio of

HPbCD contained more typical particle agglomerates and lower porosity. A semi-fractured structure was formed in some particles of C200. Data of particle size analysis was exhibited in Table 2.

Depending on the type of formulation components, their size var- ied from 6.32 to 11.37 mm with a span index ranging from 1.11 to 1.78.

Fine particle fraction and emitted dose of C30 and C300

With regard to rate constants of aggregation, the best-stabilized samples were C30 and C300. Based on their stability profile, the

Figure 1. HPLC-chromatograms of A: C10, B: C30 and C: C300.

4 S. MILANI ET AL. aerosolization potency of these samples was evaluated. The com- parison of ED and FPF for C30 and C300 is shown in Figure 5. The measured recovered doses for C30 and C300 were 93% and 87%, respectively. The ED was 93.15 and 91.23% for C30 and C300. Their respective values of FPF were 56.43 and 48.12% demonstrating higher aeorosolization efficacy of C30 over C300.

Discussion Pulmonary delivery of therapeutic proteins such as polyclonal and/or monoclonal antibodies can be suggested as an applicable method to follow both systemic as well as local treatment of dif- ferent disorders [19]. To achieve desirable shelf lives of IgG anti- bodies, drying technologies can be regarded as commonly applied methods [3,20]. SFD, as a drying technology, is capable of producing stable powders with suitable aerosol dispersion charac- ters. Unavoidable stresses inserted on protein molecules necessi- tate the incorporation of proper stabilizers [21].

Aggregation is considered as one of the most important chal- lenges for the development of stable antibody formulation as it can take place at all stages of manufacturing, shipment as well as storage time [22]. Additionally, products processed by SFD are prone to surface denaturation.

Many of the commonly utilized nonionic surfactants can well- protect proteins from surface exposure and subsequent aggregation but they may enhance the final aggregation rate due to oxidation at storage condition [23,24].

CDs have been reported as a promising category of anti-aggre- gation stabilizers. Although the capability of CDs on the protec- tion of proteins has been often attributed to binding with proteins, their role avoiding protein exposure to the water-air has also been evidenced. Besides, they are suitable candidate to manufacture engineered microparticles for respiratory delivery of peptides and proteins [25–27].

Based on the existing information in literature, the utilized ratio of CDs to proteins is in a wide range. Whether the dehydration is a dominant stress factor or surface denaturation, the respective higher and lower concentrations of CDs will be employed to serve as ideal stabilizer. As a rational approach, such a ratio should be optimized based on the protein type and the process. Although a wealth of publications, confirming the inhibition of protein aggre- gation by CDs, for antibodies as one of the most critical categories of biopharmaceuticals, few evidences are available.

Our previous study aimed to evaluate the co-application of

HPbCD and trehalose on the stability of IgG via SFD; however, the weight ratios of IgG:trehalose:HPbCD were 1:1:0.2 and 1:1:0.4 [14].

Since there is no systematic comparison dedicated to reach the exactly suitable ratio of HPbCD to IgG molecules with regards to molecular stabilization (whether acting as sugar or surfactant) and aerodynamic aspects, the current study has been proposed.

Herein, the complementary changes have been made in 2 factors.

First, the ratio of trehalose has been increased to IgG:trehalose of

1:2 (comparing the former study at which the ratio was 1:1).

Second, the HPbCD have been divided into 3 groups with approximately similar (IgG:HPbCD of 1:0.25), higher (IgG: HPbCD of

1:2) and lower (IgG: HPbCD of 1:0.05) amounts than the previous research work.

Considering aggregation as the main challenge of antibody processing and storage, the least amount of induced aggregates were observed upon storage and also the least rate of aggrega- tion by C30 and C300. The formulation C30 was composed of

IgG:trehalose:HPbCD at weight ratio 1:2:0.25 while in C300 the ratio was 1:2:0.05. The point which should be interestingly highlighted is the comparison of the aggregation profile of these formulations comparing the best sample in our previous investigation.

Regarding fully recovered monomer immediately after SFD in the mentioned study at ratio1:1:0.4 of IgG:trehalose:HPbCD, the level of induced aggregates after 1 and 3 months of storage was 2.30

Figure 2. FTIR-spectra of spray-freeze dried formulation, A:C10 and B: C300. The original and fitted trace spectra (dashed lines), the resulted fitted- curves (solid lines).

Figure 3. The result of SDS-PAGE after 2 months of storage at 45 C. From left to right, lane 1: marker; lane 2: C1; lane 3: C2; lane 4: C3; lane 5: C10; lane 6: C20; lane 7: C30; lane 8: C100; lane 9: C200; lane 10: C300.

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 5 and 2.77%, respectively. In the current research, however, calcu- lated aggregation was 0.25 ± 0.05 and 0.28 ± 0.02% even after

2 months in C30 and C300 [14].

This observation can be taken as a hint that existing differen- ces between the current formulations with the previous one have positive influence on the final stability of the product. The first change is the superior ratio of trehalose. It is clearly evident that the formulations with a low ratio of trehalose (C1, C10 and C100)

Figure 4. SEM photograph of spray freeze-dried powders at scale bar of 10mm. A:C1; B:C2; C: C3; D:C10; E: C20; F: C30; G: C100; H: C200; I: C300.

Table 2. Particle size (mm) and size-distribution index, Span, of spray-freeze dried preparations.

Formulations Mean particle size (mm) Span C1 8.45 1.22

C2 9.86 1.43 C3 11.37 1.11 C10 6.32 1.56 C20 6.98 1.78

C30 7.15 1.41 C100 7.99 1.76 C200 8.25 1.12 C300 8.46

1.75 Figure 5. Assessment of aerosol behavior of C30 and C300 including ED (%) and

FPF (%).

6 S. MILANI ET AL. were dramatically delicate to aggregation, irrespective of the ratio of HPbCD.

Comparable to previous data in the literature, for better pro- tection of antibody within storage time at high temperatures, tre- halose is the excipient of choice, even better than CDs [28,29].

The current design also approved this finding.

A variety of carbohydrates have been proved to suppress the induced-aggregation of antibodies such as rhuMAb anti-CD20, chi- meric antibody (L6) and human anti-IL8 monoclonal antibody dur- ing spray and/or freeze-drying step [30]. Among different sugars like glucose, lactose, sucrose and trehalose, two non-reducing sug- ars namely sucrose and trehalose are commonly the best choices to stabilize antibodies in the absence of water-forming hydrogen bonds with protein.

Using different carbohydrates, the sufficient quantity needs to achieve desirable stabilization depending on the type and anti- body concentration. Based on available literature outcome, anti- body stabilization against aggregation happens at a specific molar ratio of sugar:antibody which might be approximately equivalent to the capacity of antibody surface for binding to water mole- cules. Increasing trehalose to anti-HER2 antibody ratio, more than

3 folds, significantly enhanced monomer recovery of freeze-dried antibody after storage at 40 C [30].

In another study, the least suitably employed weight ratio of sucrose: IgG antibody was reported to be 2:1 to provide sufficient stabilization at 40 C. Similar to the present outputs which the best result obtained by preparations having the highest ratio of trehalose, weight ratio of 2:1 trehalose:IgG [28].

There are also some hints available about the desirable storage stability of formulations containing CDs (at different amounts) because of their high molecular weight and therefore correspond- ing high values of glass transition temperatures (Tg) as well as their amorphous nature [31,32].

Amphiphilic character of HPbCD helps well-stabilize proteins during both freezing and atomization steps of SFD. Also, HPbCD was exhibited to produce hydrogen bonds with proteins [5]. But, it remains to be evaluated at which ratio such sugar or surfactant- effect can be more influential to antibodies within SFD.

This purpose will help to correctly estimate the concentration range of HPbCD whether the surfactant effect is desired which can obtained at relatively lower concentrations or the lyoprotec- tion and hydrogen bonding is demanded which requires higher amounts. The result of the current investigation indicated that lower ratios of HPbCD to IgG (0.25 and even 0.05%) were more effective than higher amounts.

The effect of HPbCD as a surfactant found previously for lyophilized-LDH at concentrations of (0.005% to 0.1%) [31,33].

Also, HPbCD was shown to well-protect freeze-dried b-galactosi- dase (0.014%w/v) deducing the surfactant-like criteria of this derivative [34]. In another study, comparable result achieved by the combination of HPbCD (0.1%w/w) and sucrose on the stability of freeze-dried LDH [35]. On the contrary, HPbCD failed to protect

CYP3A4 at 0.003%w/v [36].

Here, higher ratio of HPbCD (more than 1:0.2 of IgG to HPbCD) turned out to be as efficient as lower amounts in IgG stabilization.

It can be hypothesized that for specific proteins with high level of solvent-exposed hydrophobic amino acid residues, CDs can exert stabilizing effect via biding to protein molecules and prevent aggregation. But, in the case of IgG within SFD, the binding between CDs and IgG might not be the main mechanism of anti- body stabilization, but the surface protection could be the major event which happened at lower amounts.

The data from FTIR were employed to estimate the secondary structure of IgG in the solid-state. All powders were conformation- ally well-protected. Trehalose can form hydrogen bonds with anti- body in the absence of water, thus protect the conformation of antibody from disturbances. It was purposed to assess the correl- ation (if any), between native structure of antibody and the con- tent of induced aggregates.

Consistent with existing data, the beta-sheet structure of all powders was in the acceptable range both after process and upon storage. Such a high amount of beta-structure was previ- ously reported in the dried-preparations of IgG, from 66% to upper levels of 85% [37,38]. It should be noted that the level of beta-structure was independent of the molecular stability of com- pounds. Thus, prevention of IgG aggregation cannot be attributed to structural preservation.

The study on the morphology of the dried particles provides information about the fundamentals of the most effective addi- tives at the best ratio to generate the suitable particle with acceptable properties for inhalation therapy. The highly porous hollow shape of particles is typical as the products of SFD.

Although the typical morphology of the particles was relatively identical, the existing difference can be attributed to the ratio of added excipients [39].

One of the crucial parameters that play an important role in the final success of the inhalable dried-powder is the aerosoliza- tion efficiency which has a definite impact on the appropriate deposition of the powders in the lung [40]. Such a porosity of powders can facilitate the flow of particles and minimize the inter-particle resistance which might be reasonably accounted for relatively high fine FPF values [41].

Application of SFD in the current study generates particles with high ED and FPF values despite their relatively large aero- dynamic diameters because of their low density that is compar- able with the existing data [39]. HPbCD, owed to its surfactant- effect, could minimize the inter-particle agglomeration resulting in enhanced aerosolization efficacy.

Comparing C30 and C300, the former demonstrated better aero- solization efficiency considering values of ED and FPF. It has been similarly demonstrated that HPbCD is capable of reducing the par- ticle sizes and enhancing the FPF [42]. It was proved that HPbCD could reduce particle sizes besides inter-particle cohesion.

Trehalose was presented to increase particle intermolecular inter- actions and lead to the formation of sticky agglomerates [43,44].

Although Trehalose has been repeatedly shown to well-stabilize proteins against surface denaturation and aggregation, the phys- ical property of the processed powders is usually poor.

Considering the tendency of such a powder is to absorb water and become moisturized, the fine powder aerosolization would be negatively influenced.

The role of anti-hygroscopicity on the enhancement of aerosol performance could also be considered. It has been previously demonstrated that HPßCD with a low-hygroscopic property, pos- sibly avoid formulation from high level of moisture uptake. Also, incorporation of HPßCD at a suitable ratio improved powder aero- solization feature. HPßCD, as a component with low density, was exhibited to promote FPF values in dry powder for inhalation [45].

In another study, addition of beta-cyclodextrin to dry powders of salbutamol prominently increased fine particle fraction of pow- ders in comparison to lactose, raffinose, trehalose and xylitol, respectively [46].

Concomitant with efficient stability, the current combination ratios of trehalose with HPbCD in C30 and C300 (FPF of 56.43 and

48.12%, respectively) offered comparable/enhanced areosolization

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY 7 in C30, comparing the previous study through which the max- imum achieved value of FPF was 51.29% [14].

Conclusion HPßCD was shown to fulfill different roles in the stabilization of proteins in almost wide range of concentrations. Employed ratios of IgG: HPbCD varied from 1:2 to 1:0.05 and it could clearly dem- onstrate that HPbCD was able to best-stabilize IgG molecules at

1:0.25 and 1:0.05 with no difference in the latter ratio. In addition to the molecular-dependent stabilization by HPbCD, the morph- ology and FPF values were also found to be influenced by formu- lation components and their amounts. Although it might be difficult to clearly attribute one mechanism of action as the dom- inant effect of IgG stabilization by HPbCD, its most-applicable range was found to be less than 1:0.25 of antibody to CD pro- vided the existence of trehalose at the ratio of IgG:sugar 1:2. From formulation aspects, it would be ideal to minimize the total added

HPbCD (from typical sugar: protein ratio of 1:1 up to 5:1 compar- ing surfactant ratio at lower concentrations); however more evalu- ations are surely recommended to mechanistically characterize the probable interactions between HPbCD and IgG via SFD and upon storage.

Disclosure statement No potential conflict of interest was reported by the author(s).

References [1] Costantino HR, Firouzabadian L, Wu C, et al. Protein spray freeze drying. 2. Effect of formulation variables on particle size and stability. J Pharm Sci. 2002;91(2):388–395. [2]

Abdul-Fattah AM, Kalonia DS, Pikal MJ. The challenge of drying method selection for protein pharmaceuticals: prod- uct quality implications. J Pharm Sci. 2007;96(8):1886–1916. [3]

Wang W. Lyophilization and development of solid protein pharmaceuticals. Int J Pharm. 2000;203(1–2):1–60. [4]

Bhatnagar BS, Bogner RH, Pikal MJ. Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol. 2007;12(5):505–523. [5]

Serno T, Geidobler R, Winter G. Protein stabilization by cyclodextrins in the liquid and dried state. Adv Drug Deliv

Rev. 2011;63(13):1086–1106. [6] Srichana T, Suedee R, Reanmongkol W. Cyclodextrin as a potential drug carrier in salbutamol dry powder aerosols: the in-vitro deposition and toxicity studies of the com- plexes. Respir Med. 2001;95(6):513–519. [7]

Dufour G, Bigazzi W, Wong N, et al. Interest of cyclodextrins in spray-dried microparticles formulation for sustained pul- monary delivery of budesonide. Int J Pharm. 2015;495(2):

869–878. [8] Tavornvipas S, Tajiri S, Hirayama F, et al. Effects of hydro- philic cyclodextrins on aggregation of recombinant human growth hormone. Pharm Res. 2004;21(12):2369–2376. [9]

Shao Z, Krishnamoorthy R, Mitra AK. Cyclodextrins as nasal absorption promoters of insulin: mechanistic evaluations.

Pharm Res. 1992;09(9):1157–1163. [10] Taneri F, G€uneri T, Aigner Z, et al. Improvement in the physicochemical properties of ketoconazole through com- plexation with cyclodextrin derivatives. J Incl Macrocycl

Chem. 2002;44(1/4):257–260. [11] Millqvist-Fureby A, Malmsten M, Bergenståhl B. Surface characterisation of freeze-dried protein/carbohydrate mix- tures. Int J Parm. 1999;191(2):103–114. [12]

Iwai J, Ogawa N, Nagase H, et al. Effects of various cyclo- dextrins on the stability of freeze-dried lactate dehydrogen- ase. J Pharm Sci. 2007;96(11):3140–3143. [13]

Carpenter JF, Pikal MJ, Chang BS, et al. Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res. 1997;14(8):969–975. [14]

Amini Pouya M, Daneshmand B, Aghababaie S, et al. Spray- freeze drying: a suitable method for aerosol delivery of antibodies in the presence of trehalose and cyclodextrins.

AAPS PharmSciTech. 2018;19(5):2247–2254. [15] Hong P, Koza S, Bouvier ES. Size-exclusion chromatography for the analysis of protein biotherapeutics and their aggre- gates.

J Liq Chromatogr Relat Technol.

2012;35(20):

2923–2950. [16] Gallagher SR. One-dimensional SDS gel electrophoresis of proteins. Curr Protoc Mol Biol. 2006;75(1):10–12. [17]

Ung KT, Rao N, Weers JG, et al. In Vitro Assessment of dose delivery performance of dry powders for inhalation. Aerosol

Sci Technol. 2014;48(10):1099–1110. [18] Schule S, Friess W, Bechtold-Peters K, et al. Conformational analysis of protein secondary structure during spray-drying of antibody/mannitol formulations. Eur J Parm Biopharm.

2007;65(1):1–9. [19] Kane C, O’Neil K, Conk M, et al. Inhalation delivery of pro- tein therapeutics. Inflamm Allergy Drug Targets. 2013;12(2):

81–87. Apr [20] Gikanga B, Turok R, Hui A, et al. Manufacturing of high-con- centration monoclonal antibody formulations via spray dry- ing-the road to manufacturing scale. PDA J Pharm Sci

Technol. 2015;69(1):59–73. [21] Sonner C, Maa YF, Lee G. Spray-freeze-drying for protein powder preparation: particle characterization and a case study with trypsinogen stability. J Pharm Sci. 2002;91(10):

2122–2139. [22] Carpenter JF, Kendrick BS, Chang BS, et al. Inhibition of stress-induced aggregation of protein therapeutics. Meth

Enzymol. 1999;309:236–255. [23] Kerwin BA. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation path- ways. J Pharm Sci. 2008;97(8):2924–2935. [24]

Wang W, Wang YJ, Wang DQ. Dual effects of Tween 80 on protein stability. Int J Pharm. 2008;347(1–2):31–38. [25]

Branchu S, Forbes RT, York P, et al. Hydroxypropyl-beta- cyclodextrin inhibits spray-drying-induced inactivation of beta-galactosidase. J Pharm Sci. 1999;88(9):905–911. [26]

Brewster ME, Hora MS, Simpkins JW, et al. Use of 2-hydrox- ypropyl-beta-cyclodextrin as a solubilizing and stabilizing excipient for protein drugs.

Pharm Res.

1991;08 (6):

792–795. [27] Castellanos IJ, Flores G, Griebenow K. Effect of cyclodextrins on alpha-chymotrypsin stability and loading in PLGA micro- spheres upon S/O/W encapsulation. J Pharm Sci. 2006;95(4):

849–858. [28] Chang L, Shepherd D, Sun J, et al. Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix? J Pharm Sci. 2005;94(7):

1427–1444. [29] Allison SD, Chang B, Randolph TW, et al. Hydrogen bonding between sugar and protein is responsible for inhibition of

8 S. MILANI ET AL. dehydration-induced protein unfolding.

Arch Biochem Biophys. 1999;365(2):289–298. [30] Wang W, Singh S, Zeng DL, et al. Antibody structure, instability, and formulation. J Pharm Sci. 2007;96(1):1–26. [31]

Anchordoquy TJ, Izutsu KI, Randolph TW, et al.

Maintenance of quaternary structure in the frozen state sta- bilizes lactate dehydrogenase during freeze-drying. Arch

Biochem Biophys. 2001;390(1):35–41. [32] Santagapita

PR, Brizuela LG, Mazzobre MF, et al.

Structure/function relationships of several biopolymers as related to invertase stability in dehydrated systems.

Biomacromolecules. 2008;9(2):741–747. [33] Yoshida A, Arima H, Uekama K, et al. Pharmaceutical evalu- ation of hydroxyalkyl ethers of b-cyclodextrins. Int J Pharm.

1988;46(3):217–222. [34] Ken-Ichi I, Sumie Y, Tadao T. Stabilization of b-galactosidase by amphiphilic additives during freeze-drying. Int J Pharm.

1993;90(3):187–194. [35] Izutsu K, Yoshioka S, Kojima S. Increased stabilizing effects of amphiphilic excipients on freeze-drying of lactate dehydrogenase (LDH) by dispersion into sugar matrices.

Pharm Res. 1995;12(6):838–843. [36] Chefson A, Zhao J, Auclair K. Sugar-mediated lyoprotection of purified human CYP3A4 and CYP2D6. J Biotechnol. 2007;

130(4):436–440. [37] Ramezani V, Vatanara A, Najafabadi AR, et al. A comparative study on the physicochemical and biological stability of

IgG1 and monoclonal antibodies during spray drying pro- cess. Daru. 2014;22(1):31–31. [38]

Griebenow K, Klibanov A. Lyophilization-Induced Reversible

Changes in the Secondary Structure of Proteins. Proc Natl

Acad Sci. 1995;92(24):10969–10976. [39] Ali ME, Lamprecht A. Spray freeze drying for dry powder inhalation of nanoparticles. Eur J Pharm Biopharm. 2014;

87(3):510–517. [40] Elversson J, Millqvist-Fureby A, Alderborn G, et al. Droplet and particle size relationship and shell thickness of inhal- able lactose particles during spray drying. J Pharm Sci.

2003;92(4):900–910. [41] Saluja V, Amorij JP, Kapteyn JC, et al. A comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation. J

Controlled Release. 2010;144(2):127–133. [42] Ramezani V, Vatanara A, Seyedabadi M, et al. Application of cyclodextrins in antibody microparticles: potentials for anti- body protection in spray drying. Drug Dev Ind Pharm.

2017;43(7):1103–1111. [43] Schule S, Schulz-Fademrecht T, Garidel P, et al. Stabilization of IgG1 in spray-dried powders for inhalation. Eur J Pharm

Biopharm. 2008;69(3):793–807. [44] Hooton JC, Jones MD, Price R. Predicting the behavior of novel sugar carriers for dry powder inhaler formulations via the use of a cohesive-adhesive force balance approach. J

Pharm Sci. 2006;95(6):1288–1297. [45] Zhao Z, Huang Z, Zhang X, et al. Low density, good flowability cyclodextrin-raffinose binary carrier for dry pow- der inhaler: anti-hygroscopicity and aerosolization perform- ance enhancement. Expert Opin Drug Deliv. 2018;15(5):

443–457. [46] Hamishehkar H, Rahimpour Y, Javadzadeh Y. The role of carrier in dry powder inhaler. Drug Discov Today. 2012;3:

39–66.

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