High-throughput formulation screening of therapeutic proteins

High-throughput formulation screening of therapeutic proteins

Drug Discovery Today: Technologies Vol. 5, No. 2–3 2008 Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Net...

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Drug Discovery Today: Technologies

Vol. 5, No. 2–3 2008

Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY

TODAY

TECHNOLOGIES

Protein therapeutics

High-throughput formulation screening of therapeutic proteins Martinus A.H. Capelle1, Tudor Arvinte1,2,* 1

Therapeomic Inc., c/o University of Geneva, Quai E-Ansermet 30, 1211 Geneva 4, Switzerland Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai E-Ansermet 30, 1211 Geneva 4, Switzerland 2

High-throughput screening (HTS) is used extensively in drug discovery to identify active compounds. Automated preparation and sample analysis in multiwell plates using a combination of liquid and/or powder handling stations, robotics and sensitive detection

Section Editors: Marco van de Weert and Eva Horn Moeller – Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark

devices provide powerful tools. At present, protein formulation remains a slow process and will benefit from a fast formulation screening approach. The use of multiwell plates enables the simultaneous screening of many excipients and experimental conditions, such as buffers, salts, surfactants, sugars, storage temperature and mechanical stress. This article reviews the application of the HTS methodology for the development of different protein formulations, such as stable liquids, lyophilisates and slow release forms.

Introduction The formulation of protein drugs is a difficult and timeconsuming process, mainly because of the complexity of protein structure and the specific physical and chemical properties involved. Practical experience has shown that there are no general approaches for proteins; each protein needs the development of a customized formulation. High-throughput screening (HTS) equipment has been continuously developing and improving over the past decade. The trend in HTS is to use less sample material, higher *Corresponding author: T. Arvinte ([email protected]) 1740-6749/$ ß 2009 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddtec.2009.03.003

density microplates, microfluidics and sensitive detection. The performance of current multiwell plate readers is comparable to classical cuvette-based spectrophotometers with the added advantage of being able to monitor the absorbance and fluorescence spectra of 96, 384 or 1536 samples in a relatively short period of time. HTS methods not only are faster in generating data, but also require new approaches in experimental design and data interpretation. The first consideration in high-throughput formulation (HTF) is the preparation of the protein formulations by dispensing excipients and the protein drug using automated liquid and/or powder handling systems. The sample analysis involves specific methods for each protein to investigate both physical and chemical characteristics of the formulations in the microplates. A continuous evaluation of the data is followed by the design of newer formulations, the process continues until a stable and active protein formulation is found. Important factors for high-throughput protein formulation are: (i) minimizing the amount of protein needed, (ii) selecting the correct assay(s) and consumables, (iii) preparing formulations using an excipient library, (iv) integrating multiple automated techniques, (v) continuous data evaluation and (vi) flexibility in designing new experiments. Therefore, a holistic approach to HTF development is required involving experimental design, method e71

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Figure 1. Protein formulation based on designing, preparing, analyzing and evaluating samples in high throughput offers a fast and detailed insight into the physical and chemical characteristics of a drug.

development, sample preparation, sample analysis, data acquisition, data evaluation and storage (Fig. 1). However, a preliminary screen is required as a guide.

Development of methods suitable for high throughput Several high-throughput approaches can be adopted to formulate drugs successfully depending on the type and characteristics of the specific protein. However, before commencing with the screening of protein formulations, methods need to be developed that can be later adapted for HTS. After establishing specific stability indicating methods, HTF screening can commence. The analytical techniques to monitor dissociation, aggregation or adsorption to surfaces should be focused on detecting changes in protein conformation and particle size. This can be achieved by screening turbidity (either nephelometry or absorbance between 320 and 450 nm), intrinsic fluorescence and the fluorescence of hydrophobic dyes. Fluorescent dyes (e.g. Nile Red [1–3], 1,8-ANS [4–6], bis-ANS [6] and Thioflavin T [6]) are used to characterize folding intermediates, measure surface hydrophobicity and detect aggregation or fibrillation [7]. These dyes are highly suitable for the highthroughput analysis of proteins on account of their versatility and sensitivity. However, in some cases the dye can affect protein conformation or stability [8]; this needs to be investigated in cuvettes before applying a dye in a HTS. Liquid chromatography and/or electrophoresis methods can be used to quantify, in a high-throughput process, the extent of protein stability. Various chromatography and electrophoresis methods need to be tested before applying a stability indicating method in a high-throughput setting. An important objective is to limit both physical and chemical degradation by optimizing the formulation.

Design of experiment: formulation approaches Formulation scientists require new ways of thinking when designing a high-throughput experiment. The use of well plates renders it possible to study the effect of many excipients and combinations. It would not be practically feasible to e72

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prepare and analyze the same number of formulations in a classical manner without the use of high-throughput methods. Different formulation approaches are reported in the literature: (i) a high-throughput combinatorial approach [9], (ii) a formulation decision tree [10], (iii) screening of one excipient type or condition at a time [11,12] or (iv) a step-by-step approach. The step-by-step approach has been successfully applied for the formulation of a malaria vaccine and for a recombinant ricin toxin A-chain vaccine [13,14] based on assessing protein stability as a function of temperature and pH, the generation of empirical phase diagrams, followed by screening of stabilizers and the ability to adsorb to aluminum salt adjuvants. Commonly used excipients in protein formulations include buffers, salts, sugars, amino acids, surfactants, antioxidants and preservatives (for a detailed list of accepted excipients for parenteral use, see Ref. [15]). Even in high throughput, it would be impossible to screen all combinations of excipients. Monitoring protein stability with different buffers and pH could seem to be a first step toward finding the best formulation. Buffer/pH screening will be discussed below in more detail for salmon calcitonin (sCT) [11] and bovine serum albumin (BSA). After the initial screening, formulation optimization depends on the specific needs of the final drug product (e.g. isotonicity, single or multi-use and protein concentration).

Design of experiment: from cuvette to microplate In 2006, the authors discussed several practical considerations for the HTF of proteins [16]. In the mean time, more experience has been gained to encourage the classical formulation scientist to transfer assays from a cuvette to a microplate. Several factors need to be taken into account when measuring absorbance or fluorescence in microplates. Many plate readers can measure either from the top or from the bottom of a well. Experience shows that generally the measurement of solutions is preferable from the bottom of a well. Measured with this orientation, fluorescence is certainly less distorted by factors such as condensation under the seal or differences in meniscus between samples. Filter- or monochromator-based fluorescence plate readers measure fluorescence intensity at a small angle (front-face fluorescence) from the bottom face of the multiwell plate. In classical cuvette-based fluorescence spectrophotometers an angle of 908 is normally used. The small angle detection of plate readers is more suitable for the analysis of turbid samples and enables the detection of fluorescence from powders. The front-face fluorescence intensity of lyophilized proteins in the solid state has been correlated with the fluorescence after reconstitution and with tertiary structural changes [17]. Another example of front-face fluorescence is the determination of the amount of riboflavin in milk [18]. The geometry of the optics in plate readers enables the characterization of proteins in gels, delivery systems and particulates [19].

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Drug Discovery Today: Technologies | Protein therapeutics

Figure 2. The volume inside a well plate was measured by determining the absorbance of water in the near infrared. A linear correlation exists between the volume or pathlength and the absorbance of water at 975 nm.

Absorbance measurements in well plates cannot directly be compared with absorbance measurements in cuvettes. In cuvettes the pathlength is fixed by the dimensions of the cuvette. In well plates, the pathlength depends on sample volume, well geometry and surface tension. However, after determining the height of a solution in a well, the effective optical pathlength is known and valid absorbance values can be measured. Assuming a cylindrical well, the volume of water in a well is proportional to the effective optical pathlength (n = pr2h) which can in turn be determined by measuring the absorbance of water at 975 nm [20]. An example of the water absorbance spectra and linear correlation between the volume-in-well and absorbance is shown in Fig. 2. The volume inside well plates needs to be controlled to ensure consistent pipetting and to monitor solution evaporation. Evaporation can occur particularly when measuring samples over long periods or at elevated temperatures. Evaporation is greater from the sides of the well plates and more pronounced when working with smaller volumes; this is called the ‘edgeeffect’ [21]. Another aspect of measuring absorbance through well plates is that all particles in the light beam are measured including those floating on the surface or sedimented to the bottom of the well. In cuvette-based measurements, the only particles taken into account are those in the light beam, in the center of the cuvette.

The surface-to-air ratio is relatively small in cuvettes but larger in 96-well plates. This difference can have an impact on the stability of proteins. The interaction of the protein with the surfaces of plastic well plates might differ from the interaction with fused-silica/glass cuvettes or primary packaging material. Quartz microplates can be used to address the influence of plastic well plates on the protein. HTS is a tool for optimizing formulation. HTF measurements do not replace the stability studies of the medication in final containers such as stoppered glass vials or prefilled ready-to-use syringes.

Sample preparation A robotic liquid handling system facilitates the routine preparation of 96- or 384-well plates containing protein formulations. Liquid handling systems have evolved over the past decade owing to the experience gained in drug discovery screening. The most important components of a highthroughput platform are the liquid handling systems in combination with one or more plate readers. Depending on the project and sample throughput required, the platform can be expanded with powder handling systems [22], robotic arms, incubators, cooling stations, pH-meters, barcode readers, stackers and shakers. The preparation of a protein formulation can consist, for example, of the mixing of three to eight solutions with microliter volumes. Typical well-volumes in 96-well plates www.drugdiscoverytoday.com

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Table 1. A list of plate reader manufacturers with their main technologies and websites Companies

Internet website

Plate reader technology

Agilent technologies

http://www.agilent.com

Lab-on-a-chip

Berthold technologies

http://www.bertholdtech.com

Filter-based plate readers

Bio-rad technologies

http://www.bio-rad.com

Filter-based plate readers

BioTek

http://www.biotek.com

Monochromator- and filter-based plate readers

BMG Labtech

http://www.bmglabtech.com

Monochromator- and filter-based plate readers Nephelometry

Caliper life sciences

http://www.caliperls.com

Lab-on-a-chip

Dynex

http://www.dynextechnologies.com

Filter-based plate readers

Edinburgh Instruments

http://www.edinst.com

Fluorescence lifetime

GE Life Sciences

http://www.gelifesciences.com

Imaging Biacore Microcal – VP capillary DSC

Hamamatsu Photonics

http://www.hamamatsu.com

Imaging

Molecular Devices

http://www.moleculardevices.com

Monochromator- and filter-based plate readers

PerkinElmer

http://www.perkinelmer.com

Monochromator- and filter-based plate readers Imaging

Tecan

http://www.tecan.com

Monochromator- and filter-based plate readers

Thermo Scientific

http://www.thermo.com

Monochromator- and filter-based plate readers Imaging

Turner Biosystems

http://www.turnerbiosystems.com

Filter-based plate readers

Wyatt technologies

http://www.wyatt.com

Dynamic light scattering

are in the range 100–300 ml; 384-well plates have wellvolumes in the range of 10–100 ml. Half diameter 96-well plates or 384-well plates are on the market for working with even lower volumes. Manually pipetting formulations into 96 wells is feasible, but difficult in 384 wells. Each formulation mixture should be reproduced preferably in triplicate to improve the significance of the data and the detection of outliers. The liquid handling systems can be programmed to pipette and mix the various solutions in the order desired. The preferred order of adding components to a well would seem to be: (i) water, (ii) buffer, (iii) other excipients (salts, sugars and preservatives among others), (iv) protein and if needed (v) dye or reagents. Predefined mixtures of excipients can be made and stored in deep well plates for later use. This will result in a decreased sample preparation time, increased sample throughput and a reduction in consumable costs. Various types of well plates are available. Black plates are most suitable for fluorescence from the top by reducing crosstalk between the wells. Transparent plates are used for absorbance assays. Cell-based assays can be performed in black or white plates with a transparent bottom. For protein formulation studies, UV transparent well plates with flat bottom are preferred allowing the monitoring of both UV absorbance and fluorescence. Other specialty plates are available, for example for crystallography, microscopy and microarray analysis. Special attachments such as the VirTis 96 well Freeze Drying System (http://www.virtis.com) have been developed to facile74

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itate the preparation of lyophilized formulations. This accessory is composed of an aluminum block that can contain 0.5– 2.0 ml vials and is designed to distribute the heat evenly to all 96 samples. Characterization of protein formulations before and after freeze-thawing or lyophilization/reconstitution will enable the selection of the best cryoprotectants and lyoprotectants.

Sample analysis: UV absorbance, fluorescence spectroscopy and microscopy Protein formulations can be analyzed in a high-throughput manner by a variety of techniques that do not require perturbance (e.g. dilution) of the formulation. An extensive list of techniques and descriptions is given in Ref. [16]. In the following sections, UV absorbance and fluorescence assays performed using plate readers are discussed. Protein concentration can be determined by measuring the UV absorbance at 280 nm [23] and used as a quality control factor in automated sample preparation. Absorbance at wavelengths higher than 320 nm is used to measure the turbidity [24]. An increase in turbidity can indicate the formation of protein aggregates. In 1999, a high-throughput turbidity assay was used by one of the authors to study conformational changes of the transforming growth factor-b3 (TGF-b3) [25]. Besides using turbidity measurements with UV–Vis absorbance plate readers, particle formation can be monitored with dedicated plate readers measuring light scattering,

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either by nephelometry (BMG Labtech, http://www.bmglabtech.com) or by dynamic light scatter (Wyatt technologies, http://www.wyatt.com). Fluorescence plate readers can be divided into two categories: filter based and monochromator based (Table 1). In general, the limit-of-detection of filter-based plate readers is lower than for monochromator-based readers. Monochromator-based readers can excite the samples and measure at specific wavelengths. The intrinsic fluorescence of the tyrosine (Tyr) and/or tryptophan (Trp) amino acids can be used to monitor local molecular changes which may also relate to global unfolding of proteins. For example, the screening of Trp fluorescence with increasing temperature has been reported as a method to relate denaturation temperature to solution condition [26]. An example is the unfolding of BSA which is known to occur at either acidic or alkaline pH values [27]. Unfolding of BSA, in a citrate buffer from pH 6 to pH 3, resulted in a blue shift of the emission maximum and a decrease in emission intensity [16]. To ascertain whether BSA unfolding depends more on pH than on buffer type, the Trp fluorescence of a hundred 2 mM BSA formulations was measured in well plates. The BSA formulations were analyzed in the same manner as described for the screening of sCT[11]. The intrinsic Trp emission of BSA was most intense at physiological pH and decreased in acidic and alkaline pH (Fig. 3), as Trp becomes more exposed and accessible to water as a result of the

Drug Discovery Today: Technologies | Protein therapeutics

unfolding BSA. In the case of BSA, the fluorescence emission depended more on the pH value than on the buffer type. Fluorescence microscopy of protein formulations stained with hydrophobic dyes (e.g. Nile Red or 1,8-ANS) can enable the detection of protein aggregates at an earlier stage than possible with standard spectroscopic and light-scatter techniques [1]. Recently, high-throughput fluorescence microscopy systems have been described with automated sample preparation, image acquisition, image analysis and data handling [28]. Combining high-throughput microscopy hardware with the fluorescence of hydrophobic dyes can result in an extra tool enabling the visualization of protein aggregate structures for the characterization of protein formulations. For the characterization of the physical stability of proteins, the following need to be monitored as a minimum: the UV absorbance spectra, intrinsic emission spectra and emission spectra of the dyes.

Sample analysis of drug delivery systems ‘Sandwich’ microplates are available that consist of a bottom compartment, which is separated from a top compartment by a specific membrane. For example, Millipore MultiscreenTM microplates (http://www.millipore.com) were developed to study drug transport across membranes. These membranes can also be used to retain a drug delivery system (e.g. microparticles) while permitting the diffusion of the released drug.

Figure 3. The intrinsic tryptophan fluorescence is shown of BSA in 100 different solutions with pH values between 2.5 and 10.5. BSA has a predominant globular heart-shaped form at physiological pH and unfolds with an increase or decrease in pH. Detailed description of buffers is in Ref. [11].

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Figure 4. The fluorescence intensity at 345 nm of BSA was measured in the top compartment of an assembled Millipore MultiscreenTM plate with a 0.65 mm PVDF membrane. BSA that permeated from the bottom to top compartment is shown in blue. BSA that permeated from the top to the bottom compartment is shown in red. For more experimental details see Ref. [19].

This high-throughput application was evaluated by studying the permeation of BSA from one compartment to another [19]. The changes in intrinsic fluorescence of BSA corresponded to the permeation of the protein from one compartment to another, as shown in Fig. 4. Ref. [11] also reports how the release of hirudin from an agar gel can be characterized simultaneously inside the gel and in the release medium. This work documents that drug delivery systems can be characterized in high-throughput using smaller sample amounts compared to the more classical drug release testing methods.

Sample analysis involving techniques of separation Size-exclusion chromatography (SEC) is often used to detect and quantify protein aggregation. Fast-SEC is a modification of conventional SEC and emerged from the field of combinatorial chemistry and HTS [29]. Analyses within 5–10 min can be achieved depending on the flow rate, column packing, length and diameter [29,30]. Other chromatographic separation methods were described for HTS [31–34]. The focus of these articles was the optimization of purification conditions by screening various columns and resins to achieve an optimal separation between an antibody and its aggregates. High-throughput hydrophobic interaction [32] and ion-exchange [34] methods can be used to quantify the degradation products of different protein formulations. The chromatography methods can detect fragments, isoforms, charge variants and deamidation products of proteins. Another recent development is the use e76

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of high-throughput self-interaction chromatography applied in combination with an artificial neural network enabling the prediction of the solubility of lysozyme in 12,636 conditions based on a small formulation screen of 81 solutions [35]. Another approach to increase the sample throughput is the use of a multichannel HPLC system [36] or a multiplexed capillary electrophoresis system [37]. One still existing challenge is to identify and quantify the oxidation and deamidation products of proteins in a highthroughput system. For this purpose, a fully automated proteolytic digestion procedure in 96-well plates has been developed by Chelius et al. [38]. Covalent modifications (e.g. methionine oxidation) of monoclonal antibodies, antibody fragments, peptide and fusion proteins were successfully identified and quantified by reversed-phase HPLC/tandem mass spectrometry [38]. However, the data processing is not fully automated and forms a bottleneck in the entire process [38]. Ultra High-Performance Liquid Chromatography (mHPLC) and X-ray diffraction are other analytical techniques that have been applied in early drug development particularly for the solubility screening of small molecules [39]. These techniques could in principle be applied for protein formulation and characterization studies, but to our knowledge no publications are available. A technique that has shown promising results for protein analysis is the lab-on-a-chip system (e.g. Labchip1 by Caliper Life Sciences, http://www.caliperls.com). These systems can detect the size of chemical or physical degradation products. The Labchip1 has been used to screen monoclonal antibody product quality and was found to be 70 times faster than a conventional capillary electrophoresis separation assay [40]. The Bioanalyzer 2100 and the LabChip Kit of Agilent Technologies (http://www.agilent.com) have been used in the detection of antibody fragments and degradation products [41]. The chip technology was found to be a valuable alternative to classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), because of (i) similar detectability but better precision, (ii) an increased sample throughput, (iii) reduced waste, (iv) simple automation and data evaluation and (v) easy to archive raw data [41]. We consider that the most promising high-throughput methods for elucidating chemical degradation products during various phases of protein drug development are: (i) fastSEC, (ii) hydrophobic interaction chromatography, (iii) ionexchange chromatography, (iv) lab-on-a-chip and (v) peptide mapping by RP-HPLC–MS–MS. Some of the discussed analytical methods might be directly applicable to protein formulation; other methods may be more challenging and require specific adaptation. Combining both physical and chemical methods to assess the stability of biologicals will assist in the search for the optimal formulation. Addition of in vitro biological activity assays to

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Drug Discovery Today: Technologies | Protein therapeutics

Figure 5. Fluorescence emission spectra of 1 mM Nile Red with 20 different formulations of 2 mg/ml salmon calcitonin are shown. The sCT formulations were dispensed in quadruplicate (rows A–D and E–H), in columns 2–11. The control solutions were dispensed in the outer columns 1 and 12. An increase in Nile Red fluorescence emission indicates a stronger binding of the dye to hydrophobic pockets that could be formed because of protein aggregation or conformational changes. Nile Red with the sCT formulations that show a low binding affinity and thus fluorescence intensity are selected for further formulation optimization.

the formulation platform will further enhance the chances of success of the protein formulation.

Evaluation of data Depending on the analytical technique, data analysis and evaluation can take longer than the data generation or sample preparation. A lot more information can be derived from measuring entire absorbance or fluorescence emission spectra, compared to measuring at a single wavelength. Monochromator-based plate readers are most suitable for this purpose. Spectroscopic data need to be interpreted without missing important information, like the position of the emission wavelength maximum in fluorescence and normalized turbidity in absorbance. Normalized turbidity is the absorbance at 350 nm divided by the absorbance at 280 nm [42], also known as the aggregation index. Proprietary software has been developed by the various manufacturers of plate readers. These computer programs could be sufficient, although problems could arise when combining information from different readers. Extracting and combining information from different types of techniques such as electrophoresis, chromatography, lab-on-a-chip, calorimetry or imaging is a challenge. In drug discovery [43], software programs such as Spotfire (http://www.spotfire.com) are used for analyzing and linking high-throughput data from

different sources. Another option is to develop visual basic macros to analyze the exported data in Microsoft Excel. Some plate readers provide data output directly in Microsoft Excel (e.g. Tecan XFluor software); others have an option to export the data. Visualization of the absorbance or fluorescence spectra in a matrix of 8  12 wells is not normally included. The authors have built a visual basic macro which allows the visualization of 96 spectra with one click of the mouse. An example of the 96 fluorescence spectra measured for the hydrophobic dye Nile Red added to different sCT formulations is shown in Fig. 5. The wells that have a strong Nile Red emission correspond to unstable formulations. The protein solutions which show no significant increase in the fluorescence of Nile Red compared to the control solutions are possible candidates for further development. Detailed information on the screening of sCT can be found in Ref. [11]. High-throughput spectroscopy was found to be a fast and sensitive method for the initial screening of the physical stability of sCT.

Conclusion In this article, different methods for the HTF of therapeutic proteins have been reviewed. HTF can be applied for both physical and chemical characterization to enable the fast development of stable peptide and protein formulations. www.drugdiscoverytoday.com

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The significant investment required to build a platform for high-throughput protein formulation will be returned by the reduced protein drug development time. Other arguments for a fast return-on-investment are the relatively small sample amounts needed, the improved protein stability and longer shelf-life of the product developed. The high number of samples per multiwell plate permits screening of the influence of multiple excipients and experimental conditions, such as, buffer type, sugars, salts, protein concentration, pH range, dosage forms, storage temperature and mechanical stress. HTS is a fast evolving technique and improved devices for detection and preparation are continuously becoming available. Upscaling the number of wells to 1536 wells with only 10 ml well volumes could further increase the throughput. The various screening tools available render large flexibility to the HTF platform. This flexibility in designing and performing experiments is essential, because no general protein stabilization approach is available. A general requirement is the optimization and adaptation of the analytical techniques to each specific protein formulation. The authors are confident that the HTF approach described in this paper will be used more and more in formulation development and will contribute in making biopharmaceuticals more stable, safe and user-friendly.

Acknowledgement We thank Dr Alex F. Drake for his crucial suggestions for improving the manuscript.

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