Thesis
Reference
Evaluation of analytical methods to address challenges in the characterization of protein formulations
PATOIS, Emilie
Abstract
Maintenir l'intégrité des protéines de la production à l'utilisation constitue un enjeu de taille pour le développement de formulations pharmaceutiques. Le besoin de méthodes de caractérisation adaptées à ces formulations va de paire. L'objet de cette thèse est d'évaluer des méthodes relativement rapides pour la caractérisation de formulations de protéines sans altération des échantillons. Différents types de produits pharmaceutiques, tels que la calcitonine de saumon, un facteur de croissance, des anticorps monoclonaux et des vaccins contre la grippe saisonnière sont étudiés. Les résultats obtenus montrent que la spectroscopie Raman de résonance dans l'ultraviolet (UVRR) avec une excitation à 244 nm est adaptée à la caractérisation des protéines dans une large gamme de concentrations et convient à l'étude des protéines en solution, agrégées ou à l'état solide. Le Nanoparticle tracking analysis (NTA) permet l'étude de l'agrégation. Des méthodes rapides permettent de caractériser la stabilité de vaccins de types sous-unitaire, fractionné ou virosomal.
PATOIS, Emilie. Evaluation of analytical methods to address challenges in the
characterization of protein formulations. Thèse de doctorat : Univ. Genève, 2011, no. Sc.
4369
URN : urn:nbn:ch:unige-220532
DOI : 10.13097/archive-ouverte/unige:22053
Available at:
http://archive-ouverte.unige.ch/unige:22053
Disclaimer: layout of this document may differ from the published version.
UNIVERSITE DE GENEVE FACULTE DES SCIENCES Section des sciences pharmaceutiques
Laboratoire de pharmacie galénique Professeur Robert Gurny Professeur Tudor Arvinte
Evaluation of Analytical Methods to Address Challenges in the Characterization of Protein Formulations
THÈSE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention interdisciplinaire
par Emilie PATOIS
de
Lons-Le-Saunier (France)
Thèse N°: 4369
Genève
Atelier de reproduction Repromail 2012
à Antonio, mon filleul
“Si nous ne faisions pas les choses dans la passion, nous ne ferions rien.”
(Henri Millon de Montherlant, Le Cardinal d’Espagne).
Table of Contents
Introduction ... 1
Chapter I Ultraviolet resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch-peptide conjugate and TGF-β3... 7
Chapter II Stability of seasonal influenza vaccines investigated by spectroscopy and microscopy methods... 31
Chapter III Evaluation of nanoparticle tracking analysis (NTA) in the characterization of therapeutic antibodies and seasonal influenza vaccines... 61
Chapter IV Studies of influenza vaccines by nanoparticle tracking analysis, spectroscopy and microscopy techniques... 99
Conclusion and Perspectives ... 137
Résumé (French summary) ... 141
Remerciements (Acknowledgements) ... 145
Abbreviations... 147
Introduction
Proteins consist of unbranched polypeptide chains made from the 20 genetically encoded amino acids. The understanding of proteins has continued to improve with the development of new technologies and characterization techniques since the early elementary analyses of egg and serum albumins based on their sulfur and phosphorus content in 1819. At the beginning of the XXth century, the development of methods went along with discovering proteins isolated from biological systems and brought an understanding of their composition and functions [1-4]. It has always been acknowledged that the understanding protein behavior requires the study of complementary parameters and the characterization of proteins in their environment [5]. Technological advances of the XXth century resulted in more detailed characterization of proteins and the appearance of new classes of pharmaceutical products based on hormones, enzymes, blood factors, cytokines, vaccines and monoclonal antibodies [6]. In the last 25 years, more than 130 therapeutic proteins have been approved for a wide range of pharmacological indications [7]. Proteins, which play a variety of roles in the body, represent a growing area in the pharmaceutical field.
A challenging task in the development of protein-based pharmaceuticals is the maintenance of safety and efficacy throughout the lifetime of the product. These issues are all the more important as a wide range of factors can affect protein conformation, structure and activity at any stage from the production phase to the end use [8-13]. The study of formulated proteins requires methods adapted to final drug products and the ability to detect subtle changes [10, 14, 15]. The present work focuses on the characterization of protein formulations by relatively fast analytical methods with no or only minor sample preparation. The results presented were mainly obtained by spectroscopy techniques, which have the advantage of
providing information based on the interaction of electromagnetic radiation with proteins without affecting their environment.
The first part of this thesis cnocerns UV resonance Raman spectroscopy (UVRR) with excitation at 244 nm as a tool for rapid analysis of final protein formulations. This technique is based on the study of vibrations specific to aromatic amino acids and amide bonds. UVRR has already been shown to provide details about the structure of proteins and on the effect of their environment in studies, where spectra were recorded over 10 minutes to 2 hours [16-19].
Technical improvements have allowed faster measurements. In this work, spectra were recorded in 5 minutes. Such fast acquisition times will enable UVRR to become a valuable complement to available spectroscopic techniques for the characterization of protein structure from research to quality control.
The second part of this thesis evaluates the use of optical spectroscopy and microscopy techniques, currently used to study therapeutic proteins, as a new approach to monitor the stability of vaccines. Subunit, split and virosomal seasonal influenza vaccines administered in the 2009-2010 season are studied. While the stability of pharmaceuticals can be defined as “the capability of a particular formulation in a specific container / closure system to remain within its physical, chemical, microbiological, therapeutic, and toxicological specifications” [20], stability studies of vaccines are mainly based on biological activity assays [9]. Such assays are expensive and time-consuming. Fast analytical methods to characterize the stability of the physico chemical properties of vaccines could complement biological assays. Fluorescence emission was used to detect changes in the environment of chromophores present in the active pharmaceutical ingredient of influenza vaccines. Methods for the detection of aggregation were also investigated such as static light scattering and Nile Red fluorescence microscopy. These orthogonal methods allow the definition of different
stability profiles for vaccines produced by different manufacturers. Such characterization tools will benefit the development and production processes of such complex protein formulations.
The third part of this thesis describes nanoparticle tracking analysis (NTA) as a potentially useful tool for the characterization of monoclonal antibodies (mAb) and seasonal influenza vaccines. NTA is a relatively new technique for size characterization of particles between 40 and 1000 nm. The detection and characterization of protein aggregates in this size range remains a major issue for the development and use of protein pharmaceuticals [21-28].
NTA has the advantage of being suitable for polydispersed samples [29] to complement other techniques [30-32]. With NTA, video recordings are first captured and then analyzed.
Visualization of the Brownian motion of particles on video recordings can provide new real-time insights into protein aggregation.
In the fourth part of this thesis, NTA, spectroscopy and microscopy techniques are used to study the stability of seasonal influenza vaccines of the 2010-2011 season. Updated versions of subunit, split and virosomal vaccines analyzed in the second part are studied.
Depending on the vaccine, aggregation was induced by temperature stress or a freeze-thaw cycle. The proposed approach, based on orthogonal techniques, may be advantageously used to study the stability of vaccines.
The overall aim of this thesis is to describe the evaluation of orthogonal analytical methods for the characterization of protein formulations. Such tools will help understanding relevant issues such as variations of production processes.
References
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Chapter I
Ultraviolet resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch-peptide conjugate and TGF- β 3
E. Patois1, I.A. Larmour2, S.E.J. Bell2, C. Palais3, M.A.H. Capelle3, R. Gurny1 and T. Arvinte1,3
1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest Ansermet 30, 1211 Geneva, Switzerland
2 Innovative Molecular Materials Group, School of Chemistry and Chemical Engineering, Queen’s University of Belfast, Belfast, BT9 5AG, United Kingdom
3 Therapeomic Inc., WRO-1055, Mattenstrasse 22, 4002 Basel, Switzerland
Eur J Pharm Biopharm 81 (2), June 2012, 392–398 Abstract
Ultraviolet Resonance Raman (UVRR) spectroscopy with excitation at 244 nm was investigated here as a possible useful tool for fast characterization of biopharmaceuticals.
Studies were performed on three protein drugs: salmon calcitonin (sCT), starch-peptide conjugate and transforming growth factor-β3 (TGF-β3) adsorbed onto solid granules of tricalcium phosphate (TCP). Secondary structure of sCT was investigated for solutions of 0.5 mg/mL up to 200 mg/mL, regardless of the turbidity or aggregation states. An increase in β-sheet content was detected when sCT solutions aggregated. UVRR spectroscopy also detected small amount of residual organic solvent in a starch-peptide conjugate solution containing only 40 µg/mL of peptide. UVRR spectroscopy was then used to characterize a protein, TGF-β3, adsorbed onto solid granules of TCP at 50 and 250 µg/cm3. This study
shows that UVRR is suitable to characterize the protein formulations in a broad range of concentrations, in liquid, aggregated and solid states.
1. Introduction
The development and marketability of biopharmaceuticals requires formulations to maintain their native structure and activity throughout their lifetime. There is an unmet need for complementary analytical methods, which can detect subtle changes in the structure of proteins within formulations and can easily detect possible degradations of materials [1].
Ultraviolet Resonance Raman (UVRR) spectroscopy can complement available spectroscopic techniques, such as infrared absorption (IR) and circular dichroism (CD), for protein characterization during the development as well as for product quality control.
UVRR spectral features provide information on the structure of proteins. Depending on the excitation wavelength, UVRR spectra refer to vibrations of amides [2] and/or aromatic components [2-5]. Excitation around 200 nm enhances amide vibrations [6].
Amide bands I, II and III, sensitive to protein conformation, are used to indicate the backbone secondary structure [6-10]. UVRR spectroscopy also detects an amide band, referred to as amide S, which is related to all non-α-helical structures [11]. Excitation wavelengths from 220 to 250 nm favor the enhancement of aromatic vibrations [3, 6, 12], which provide information on the local environment of tryptophan, tyrosine and phenylalanine aromatic residues [2, 12, 13]. Doublets of peaks located at 850 / 830 cm-1 and 1360 / 1340 cm-1 correspond to Fermi doublets of tyrosine and tryptophan, respectively [2-5, 12-14]. The relative intensity ratio I(850)/I(830) reflects the H-bonding status of the tyrosine hydroxyl group [2, 14]. The relative intensity ratio I(1360)/I(1340) reflects the hydrophobicity / hydrophilicity of the environment of the tryptophan indole group [2, 12].
UVRR spectroscopy has been widely applied to proteins within academic research.
Since the mid-1990s, a range of experimental systems have been designed [13, 15, 16] and used to study viruses assembled with DNA and proteins [17-20], structural changes associated with redox and ligation changes of cytochrome c [16], protein interactions with oxygen [21], membrane proteins [22, 23], peptide-vesicle interactions [24], among others. These studies have provided new insight into the detailed structure of proteins and the effect of their environment.
The underpinning academic research indicates that UVRR spectroscopy is a powerful tool for research studies, where the experimental conditions, such as sample medium and acquisition time (which can range from 10 min to 2 h), can be chosen without compromise to maximize the signal quality. In addition, detailed and exhaustive quantitative analyses are used to extract the maximum amount of information from UVRR spectra. The aim of this study was to investigate the use of UVRR spectroscopy with excitation at 244 nm as a tool for rapid analysis of final protein formulations, with an acquisition time of 5 min and without exhaustive quantitative analysis. Among the wavelengths used for protein studies with UVRR spectroscopy, 244 nm is the only one widely available outside specialist laboratories.
Excitation at 244 nm has already been shown to allow simultaneous studies of aromatic amino acids and deoxynucleosides [25], as well as the detection of some amide bands [26]. In addition, the use of this wavelength allows the proteins to be studied in the presence of oxygen, whose low absorption above 195 nm results in it giving only a minor contribution to the recorded spectra [27-29]. The oxygen band, which may overlap with the amide II band [30], can therefore be easily subtracted together with the background by using a solvent blank.
This made the wavelength of 244 nm the natural choice for the current exploratory studies.
In the present study, three protein drugs were investigated in similar conditions to their final formulations. The samples were chosen to be a representative of the broad range of sample types that may be encountered. Solutions of salmon calcitonin (sCT) in water were characterized over a broad range of concentrations, from 0.5 to 200 mg/mL.
Conformational changes of 5 mg/mL sCT were investigated in six buffer solutions at pH 5 or pH 6 over 24 h at room temperature. A starch-peptide conjugate containing 40 µg/mL of peptide was characterized before and after lyophilization to evaluate whether UVRR spectroscopy could explain a stability difference. Solid forms of transforming growth factor-β3 (TGF-β3) adsorbed onto granules of tricalcium phosphate (TCP) were studied as an example of solid biopharmaceuticals characterization using UVRR.
2. Materials and Methods 2.1 Materials
Buffer solutions at 20 mM were prepared from chemicals purchased from Fluka AG (Buchs, Switzerland) or Riedel de Haën GmbH (Seelze, Germany). The following buffers at pH 5 were used in this experiment: acetic acid-sodium acetate, citric acid-sodium citrate and citric acid-sodium phosphate dibasic. The following buffers at pH 6 were used in this experiment: citric acid-sodium citrate, citric acid-sodium phosphate dibasic and sodium phosphate monobasic-sodium phosphate dibasic. sCT and TGF-β3 adsorbed onto TCP granules were obtained from Novartis AG (Basel, Switzerland) and Therapeomic Inc.
(Basel, Switzerland), respectively. Solution and lyophilized form of starch-peptide conjugate and lyophilized starch were provided by Aplagen GmbH (Baesweiler, Germany).
2.2 Preparation of protein formulations
sCT was dissolved in water at 10, 50 and 200 mg/mL. Solutions at 0.5, 1, 2 and 5 mg/mL of sCT were obtained from dilution of the 10 mg/mL sCT solution. sCT solution at 20 mg/mL was diluted from a 50 mg/mL sCT solution. Solutions at 5 mg/mL sCT in 10 mM buffers were obtained by a twofold dilution of 10 mg/mL sCT in 20 mM buffers.
Lyophilized forms of starch-peptide conjugate and starch were reconstituted with water just prior to measurements. Solution of starch-peptide conjugate was used as provided.
Samples of TGF-β3 adsorbed onto TCP granules at 10, 50 and 250 µg/cm3 of TGF-β3 were measured without any preparation.
2.3 UVRR spectroscopy
Biopharmaceutical formulations were analyzed in 0.5 mL disposable sample holders, which were prepared by removing the top section from 2 mL Eppendorf tubes. These were a standard size and thus allowed rapid interchange between samples (20 s between successive measurements). Fresh holders were used for each sample, which minimized cross- contamination. Irradiation of the surface of the liquid minimized interfering Raman scattering signals from the container.
UVRR spectra were measured over a spectral range of 325-2230 cm-1 with 2 cm-1 resolution and excitation at 244 nm, which was generated by frequency doubling of an Innova FRED 300c Ar+ laser. The power of the fundamental was locked at 120 mW, which resulted in 26 mW of 244 nm at the sample. A Spex Triplemate spectrograph (filter stage:
1200 grooves/mm, spectrograph: 3600 grooves/mm) was used along with an Andor DU420A CCD camera (1024 × 255) at -80°C as detector. The setup allowed samples to be changed within 20 s by flipping the Ar+ mirror mount out of the beam path (Figure 1).
During measurement, the system was rotating at 58 rpm. The focus point was set at the surface of the samples.
Data acquisition was carried out by the accumulation of Raman signals in kinetic mode over 5 min total integration with readouts every minute. The spectra reported here are the sums of the kinetic runs. Spectra of sCT were normalized to the band at 1615 cm-1 to compensate for changes in focus/laser power between samples.
3. Results
UVRR spectroscopy detects signals arising from amide backbone as well as from aromatic side chains. The three protein drugs, which were investigated, contained the following aromatic residues: sCT, one tyrosine, starch-peptide conjugate, tyrosine and phenylalanine residues and TGF-β3, tryptophan, tyrosine and phenylalanine residues. The conjugated peptide also had a naphthalene group detectable with UVRR spectroscopy. All
Figure 1. Sample holder setup.
investigated materials contained cysteine amino acids, whose SH, CS or SS bond vibrations can also be detected with UVRR spectroscopy [2]. It has to be noted that bands characteristic of free sulfhydryl groups, SH bands located between 2500 and 2700 cm-1, as well as SS bands characteristic of disulfide groups, located between 500 and 550 cm-1, were outside the detection range of our spectrometer.
3.1 sCT solutions
Spectrum of sCT obtained from a 5 mg/mL sCT formulation in water after subtraction of the water background (Figure 2) has twelve identifiable bands, which are described in Table 1. The majority are tyrosine vibrations, which were preferentially enhanced with excitation at 244 nm. The spectrum is also composed of amide bands, a vibration specific to
Figure 2. UVRR vibration bands detected for sCT at 5 mg/mL in water.
non-aromatic side chains and a cysteine vibration. The tyrosine vibrations provide information on the tyrosine environment. Vibrations assigned to the tyrosine residue of sCT were at 1615 cm-1, the strongly enhanced ν8a mode derivative of benzene vibration;
at 1178 cm-1, an in-plane CH deformation; at 1210 cm-1, a symmetric ring stretching commonly observed in para-substituted benzene derivatives; another ring stretching [31]
at 640 cm-1; and a doublet of low intensity peaks at 830 and 850 cm-1. The latter results from a Fermi resonance between the symmetric ring breathing fundamental (νl) and the overtone of an out-of-plane C-H bend of all symmetry ring deformation mode (ν16). The observed relative intensity ratio I(850)/I(830) lay within the range associated with the phenolic OH group of tyrosine acting simultaneously as an acceptor and donor of moderate to weak H-bonds [2, 14].
Secondary structure of the polypeptide backbone can be evaluated from the resonance-enhanced amide bands. The amide I band, related to C=O stretching, has a peak position reflecting H-bond donation to C=O groups [2, 7]. Amide II and amide III bands relate to C-N stretching and in plane N-H bending combined in- and out-of-phase, respectively. The amide III bands are sensitive to secondary structure [2, 7]. In the sCT spectrum, as shown in Figure 2, amide I, II and III bands are located at 1677, 1555 and in the 1250-1350 cm-1 region. The amide I band at 1677 cm-1 was assigned to β-sheet.
The band at 1555 cm-1 was assigned to amide II [6, 7, 10, 11] since the aromatic amino acids present in sCT had no strong bands in this spectral region [2], although other proteins that contained tryptophan had also an interfering tryptophan band at 1550 cm-1. The amide III bands are at 1258 cm-1, a characteristic band of unordered structure and around 1330 cm-1, where less intense bands were assigned to α-helical content. The C-H
bending band detected in the 1380 cm-1 region was specific to non-α-helical structures [11].
The cysteine vibration located at 705 cm-1 was characteristic of the intramolecular disulfide bond of sCT [2].
Table 1. Spectral assignments and structure correlations of sCT UVRR bands.
Raman shift
(cm-1) Description Secondary Structure
1615 Tyrosine [2, 7, 25] ν8a benzene vibration 1210 Tyrosine [2, 7] ν7a symmetric ring stretch of
para-substituted benzene 1178 Tyrosine [2, 7] ν9a in-plane CH bend,
C6H5-C stretch 850 / 830 Tyrosine [2, 14]
Fermi resonance doublet ν1 / 2ν16a
(H-bonding effect)
640 Tyrosine [31] ring stretch
1458 Non aromatic
side chain [26] CH2 deformation 705 Cysteine [2] C-S stretch of trans
conformer
1677 Amide I [2, 7] H-bonded C=O stretch β-sheet and β-barrel 1555 Amide II [6, 7, 10, 11] C-N stretch and N-H
bending All
1330 Amide III [2, 7] N-H and C-H bend α-helix
1258 Amide III [2, 7, 10] N-H and C-H bend disordered
3.1.1 Effect of concentration
Solutions of sCT in water were characterized by UVRR spectroscopy over a wide range of concentrations, from 0.5 to 200 mg/mL. The normalized spectra (Figure 3) described a similar secondary structure composed of an unordered part, β-sheet and a minor α-helical content. At 200 mg/mL, sCT solution turned from a transparent solution to a turbid gel and the amide I band characteristic of β-sheet (1677cm-1) increased.
Figure 3. UVRR spectra of sCT in water at different concentrations.
3.1.2 Stability study during 24 hours at room temperature
Spectra of 5 mg/mL sCT in a series of 10mM buffers at pH 5 and pH 6 were recorded at several time points over 24 h. UVRR spectra of all buffer solutions were collected and subtracted from sCT spectra. A solution of 5 mg/mL sCT in water was used as a control.
Spectra measured a few minutes after solution preparation and after 24 h were compared (Figure 4). For most of the buffers, the spectra were similar to spectra of sCT in water and remained unchanged after 24 h. Only in the citric acid-sodium citrate at buffer pH 5, an increase in β-sheet content was observed in the amide regions (Figure 4).
Figure 4. UVRR spectra recorded after preparation (A) and after 24 h at room temperature (B) for sCT at 5 mg/mL in 10 mM buffers at pH 6 (a-c), at pH 5 (d-f) and in water (g). (a and d) citric acid-sodium phosphate dibasic, (b) sodium phosphate monobasic-sodium phosphate dibasic, (c and f) citric acid-sodium citrate and (e) acetic acid-sodium acetate.
Figure 5 shows the complete sequence of spectra measured from time 0 to 24 h for sCT in citric acid-sodium citrate buffer at pH 5. The two spectra measured after 12 and 24 h show an increase in the β-sheet content with time, as observed in both amide I and III regions.
This conformation change was concomitant with the appearance of small white aggregates in suspension.
Figure 5. UVRR spectra recorded after preparation and after 3, 6, 9, 12 and 24 h incubation at room temperature for sCT at 5 mg/mL in 10 mM citric acid-sodium citrate buffer at pH 5.
3.2 Starch-peptide conjugate solutions
A solution of starch-peptide conjugate containing 40 µg/mL of peptide was characterized before and after lyophilization (Figure 6a and b). Starch solution (Figure 6c) and water (Figure 6d) were used as controls.
Broad background features dominate the four spectra. A contribution from oxygen at 1555 cm-1 could be detected on the background spectrum. The band was weak in comparison with water vibration at 1650 cm-1, similar to the study of protein solution performed with excitation at 204 nm [32]. UVRR spectra of the conjugate solutions after subtraction of the starch contribution were compared with the starch spectrum obtained after the subtraction of water from the starch solution (Figure 7). Between 800 and 1800 cm-1, seven vibration bands were detected for the starch-peptide conjugate solution (Table 2).
Figure 6. UVRR spectra recorded for aqueous solutions of (a) starch- peptide conjugate, (b) reconstituted lyophilized form of the conjugate, (c) starch and (d) the water background.
Table 2. Spectral assignments of UVRR bands detected for starch-peptide conjugate solution.
Bands detected in the reconstituted lyophilized form are presented in the third column.
Raman shift
(cm-1) Description Detection in the reconstituted
lyophilized form
1626 Tyrosine or naphthalene yes
1453 Ethanol no
1374 Naphthalene yes
1278 Ethanol no
1083 Ethanol no
1045 Ethanol or naphthalene no
879 Ethanol or naphthalene no
Figure 7. UVRR spectra recorded for aqueous solutions of (a) starch- peptide conjugate, (b) reconstituted lyophilized form of the conjugate and (c) starch (after subtraction of the starch signal for (a) and (b) and of the water signal for (c)).
Of these seven bands, only those at 1626 and 1374 cm-1 were detected for the reconstituted lyophilized form. The 1626 cm-1 was assigned to tyrosine ring stretching mode [5], although naphthalene CC stretching [33] may also contribute to this band. The absence of strong bands around 1000 cm-1, which would be a characteristic of phenylalanine, suggested that the phenylalanine contribution to this spectrum was minimal. The 1374 cm-1 band was assigned to naphthalene CH wagging. Three of the five additional bands of the starch-peptide conjugate solution at 879, 1278 and 1453 cm-1 were assigned to residual ethanol from manufacturing. The bands at 1045 and 1083 cm-1 could be assigned either to ethanol or naphthalene [34]. Their relative intensity with respect to the other ethanol bands suggested that they were predominantly due to ethanol. The comparison of spectra of the starch-peptide conjugate solution and its reconstituted lyophilized form shows that traces of organic solvent remained after purification.
3.3 TGF-β3 adsorbed onto TCP granules
Identification of protein-related vibration bands in solid samples of TGF-β3 adsorbed onto TCP at 10, 50 and 250 µg/cm3 was studied by UVRR spectroscopy. Figure 8 shows the raw data of the TGF-β3 sample at 250 µg/cm3 and TCP. The strong TCP bands located around 950 cm-1 dominate both spectra. An additional band at 1614 cm-1 was detected in the sample containing TGF-β3. Subtraction of the TCP background allowed other bands to be discerned and left only a residual noise-like feature at the position of the strong TCP band (Figure 9). No protein band was detected from the sample at 10 µg/cm3 TGF-β3. Samples at 50 and 250 µg/cm3 TGF-β3 produced UVRR spectra with vibration bands at 1614 cm-1 and 1550 cm-1. The band at 1614 cm-1 corresponds to aromatic amino acid ring breathing [2, 4].
Tryptophan, phenylalanine and tyrosine side chains may contribute to this vibrational mode.
The second band at 1550 cm-1 arose from in-plane ring stretching of tryptophan [4] and/or
amide II vibrations [10]. The relative heights of the TGF-β3 bands, relative to those of TCP, were calculated. A ratio of 1:4.9 was obtained from the normalized protein band intensities calculated for the 50 and 250 µg/cm3 samples.
Figure 9. UVRR spectra measured from solid samples of (a) 250 µg/cm3, (b) 50 µg/cm3 and (c) 10 µg/cm3 TGF-β3 adsorbed onto TCP (after subtraction of TCP contribution, which leaves a residual noise-like feature at around 950 cm-1).
Figure 8. UVRR spectra measured from solid samples of (a) 250 µg/cm3 of TGF-β3 adsorbed onto TCP and (b) TCP.
4. Discussion
sCT has been used as a therapeutic agent for metabolic bone disease for more than 30 years [35]. Previous studies performed with spectroscopic techniques, such as circular dichroism, infrared spectroscopy, and fluorescence emission, showed that calcitonin has a largely unordered structure in aqueous media [36, 37]. To our knowledge, the UVRR spectrum of sCT is reported for the first time in this paper. UVRR spectroscopy of sCT provided a clear sensitivity advantage over IR absorption since the spectra were recorded over a wide concentration range, from 0.5 mg/mL up to 200 mg/mL, without any change in the experimental conditions. The lowest concentration in these experiments, 0.5 mg/mL sCT, was below the limit of detection reported for IR absorption experiments, 3-5 mg/mL [38]. The intensity of tyrosine bands in sCT UVRR spectra was unexpected considering that sCT contains 32 amino acids, only one of which is tyrosine. Indeed, total tyrosine concentration in formulations was approximately 30× lower than the overall protein concentration, which showed that UVRR spectroscopy is a highly sensitive method.
At all the concentrations studied, UVRR spectra showed that sCT formulations contained an unordered structure (amide III), minor contents of α-helix (amide III) and β-sheet (amide I). It has to be noted that amide III bands, which were not commonly used to evaluate the secondary structure of proteins due to their low signal relatively to other amide bands, have become an indicator of secondary structure of proteins, since they allow better understanding of folding [39, 40] and more particularly since the frequency dependence of amide III vibrations on the Ψ Ramachandran angle has been shown [41, 42]. These results complemented the information derived from other spectroscopic techniques. At 200 mg/mL, the solution of sCT in water formed a turbid gel and UVRR spectroscopy detected
an increase in β-sheet content in the amide I region. To our knowledge, it is the first time that an increase in β-sheet content is reported in the case of sCT gel, even though it has been observed for human calcitonin [36, 37]. UVRR spectroscopy proved to be suitable for studying the secondary structure of protein over a wide concentration range, which permitted easy detection of this conformation change.
The study of sCT stability during 24 h showed that spectral measurements were reproducible over extended periods of time. This reproducibility is essential if changes in physical state, which give rise to relatively small changes in spectral features, are to be detected. For example, the data in Figure 4 show that there were no significant differences between UVRR spectra of sCT recorded at early time and after 24 h for all but one formulation. Even the subtle differences in UVRR spectra recorded at early time between formulations were retained after 24 hours. This demonstrated that the small differences between UVRR spectra were real features of the formulations, rather than arising from random experimental factors. During the 24 h stability study of sCT solutions, the β-sheet content increased with time for the formulation in 10mM citric acid-sodium citrate buffer at pH 5 (Figure 4 and Figure 5). This increase in β-sheet content occurred during the formation of aggregates in suspension visible by eye.
The second active compound investigated in this study was a starch-peptide conjugate containing 40 µg/mL of peptide. Stability of this conjugate is enhanced after lyophilization and reconstitution in water (Arvinte et al., unpublished data). Both the starch-peptide conjugate solution and its lyophilized form reconstituted with water were analyzed by UVRR spectroscopy. As shown in Figure 7, UVRR spectra measured for the starch-peptide conjugate solution and its reconstituted lyophilized form differed by the presence/absence of five ethanol peaks on top of the peptide bands. The presence of these peaks in the starch-peptide
solution, before lyophilization, indicated the presence of traces of organic solvent. After lyophilization, these traces were not observed. This confirmed the assignment hypothesis made for two peaks, which might have been assigned to naphthalene. Given the destabilizing effect that organic solvents can have on proteins, it is possible that the traces of ethanol were related to the instability of the peptide-starch solution before lyophilization. These results showed the importance of characterizing biopharmaceuticals in their final formulations. The sensitivity of UVRR spectroscopy for the starch-peptide conjugate was 40 µg/mL peptide.
This concentration was about 10× lower than for sCT formulations discussed previously.
UVRR spectroscopy was also used to investigate a solid formulation consisting of the growth factor TGF-β3, adsorbed onto a tricalcium phosphate (TCP) scaffold. Such a system, combining the osteoinductive properties of TGF-β3 with the osteoconductive properties of TCP, is of interest in the field of bone healing [43]. UVRR spectra recorded for formulations containing 50 and 250 µg/cm3 of TGF-β3 adsorbed onto TCP permitted the identification of aromatic vibrational bands of TGF-β3. This was possible although the TCP matrix had a strong signal, which could be subtracted. UVRR spectra measured from samples at 50 and 250 µg/cm3 TGF-β3 only differed by the bands’ height. The intensity measured at 1614 cm-1 for the 250 µg/cm3 TGF-β3 sample was about five times more intense than the same band for the 50 µg/cm3 sample, indicating that UVRR spectroscopy may be used for quantitative measurements of protein adsorbed onto TCP. These results showed the potential use of UVRR spectroscopy to characterize the protein in solid state.
5. Conclusion
UVRR spectroscopy was applied to characterize three protein drugs in different formulations. Signals related to the aromatic amino acids and the amide bands were detected
using excitation at 244 nm. In sCT solutions from 0.5 to 200 mg/mL, UVRR spectroscopy detected the presence of unordered structure, minor contents of α-helix and β-sheet, regardless of the turbidity or aggregation state. An increase in β-sheet content was observed when sCT solution turned from a transparent solution to a turbid gel at 200 mg/mL and during aggregation in 10 mM citric acid-sodium citrate buffer at pH 5. UVRR spectroscopy proved to be very sensitive in the detection of trace amounts of ethanol in a solution containing a starch-peptide conjugate at a concentration of 40 µg/mL peptide. UVRR spectroscopy can detect TGF-β3 adsorbed onto solid TCP granules at 50 and 250 µg/cm3. These examples show that UVRR spectroscopy can be used for the characterization of a large variety of protein formulations, such as solids, liquids, gels or suspensions of aggregates, without the need of specific sample preparation.
Acknowledgements
The authors acknowledge Sue Tavender from Rutherford Appleton Laboratories (Didcot, United Kingdom) for her technical assistance and the company Aplagen GmbH (Baesweiler, Germany) for providing the starch and starch-peptide conjugate samples. We also thank the Science and Technology Facilities Council for access to the Lasers for Science Facility in Rutherford Appleton Laboratories (Project Number 72004).
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Chapter II
Stability of seasonal influenza vaccines investigated by spectroscopy and microscopy methods
E. Patois1, M.A.H. Capelle2, R. Gurny1 and T. Arvinte1,2
1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest Ansermet 30, 1211 Geneva, Switzerland
2 Therapeomic Inc., WRO-1055, Mattenstrasse 22, 4002 Basel, Switzerland
Vaccine 2011, 29(43), 7404-13 Abstract
The stability of different seasonal influenza vaccines was investigated by spectroscopy and microscopy methods before and after the following stress-conditions: i) 2 and 4 weeks storage at 25°C, ii) 1 day storage at 37°C and iii) one freeze-thaw cycle. The subunit vaccine Influvac® (Solvay Pharma) and the split vaccine Mutagrip® (Sanofi Pasteur) were affected by all stresses. The split vaccine Fluarix® (GlaxoSmithKline) was affected only by storage at 25°C. The virosomal vaccine Inflexal® V (Berna Biotech) was stable after the temperature stresses but aggregated after one freeze-thaw cycle. This study provides new insights into commercial vaccines of low antigen concentration and highlights the importance of using multiple techniques to assess vaccine stability.
1. Introduction
Vaccination remains the most effective method to protect against infectious diseases such as influenza [1, 2]. The poor stability of vaccines is a hurdle to their development and use [2, 3], an increase in the temperature of storage or freezing might be detrimental to influenza vaccines [4, 5]. At present the characterization of stability is mainly based on biological activity assays [3, 6]. Fast analytical tools to characterize the physical and chemical properties could contribute to better understanding of critical stability issues. In the field of recombinant proteins, extensive physical and chemical stability studies are performed to assure the quality of the products [7-11]. There is an interest in applying such studies to vaccines, which consist of complex protein formulations containing, beside the antigenic proteins, residual viral proteins and lipids, preservatives, antiseptic agents, inactivating agents, excipients and residues from cells or eggs depending on the manufacturing process.
The complexity of subunit and split vaccines is demonstrated in the electron microscopy and chromatography study performed by Renfrey and Watts [12]. Methods to study such complex formulations need to detect changes in antigenic protein structure and aggregation. In recent years, different methods have been used to study vaccines; these include differential scanning calorimetry, nuclear magnetic resonance spectroscopy, mass spectrometry, field flow fractionation, two-dimensional high-performance liquid chromatography and size-exclusion chromatography [13-19].
In the present study, spectroscopy and microscopy methods are applied to study the stability of trivalent vaccines available on the market in Europe during the 2009-2010 influenza season: (a) Mutagrip® (Sanofi Pasteur), (b) Inflexal® V (Berna Biotech), (c) Influvac® (Solvay Pharma) and (d) Fluarix® (GSK). These vaccines, produced from inactivated influenza virus grown in embryonated chicken eggs, do not contain any adjuvant
and are clear by eye. Vaccines (a) and (d) are split vaccines, based on disrupted virus particles [2], (c) a subunit vaccine, containing only virus surface antigens, neuraminidase and hemagglutinin, and (b) a virosomal vaccine, in which virus surface antigens were integrated into phosphatidylcholine bilayer liposomes with an average diameter of 150 nm [20, 21].
Spectroscopy and microscopy methods provide information on structural changes as well as aggregation, and indirectly on the stability of complex protein formulations. Static light scattering at 90° measured with spectro-fluorimeters is very sensitive in detecting changes in protein aggregation [22-24] and for the characterization of antigens bound to aluminium adjuvants [25]. Intrinsic fluorescence is widely used to study conformational changes of proteins, since fluorescent amino acids, mainly tryptophan and tyrosine, can be excited within complex formulations [26, 27]. Measurement of fluorescence lifetime gives insight into the mobility of tryptophan residues [28]. Tryptophan fluorescence emission has already been applied to characterize protein aggregation [24, 29, 30], thermal stability of adenovirus [31] or the stabilizing effect of excipients on virus like particles [32]. Fluorescence microscopy with Nile Red staining is used to detect large protein aggregates, from half a micrometer to several millimeters in diameter [24, 33]. The hydrophobic dye, Nile Red, allows detection of aggregates while minimizing alterations to the local environment of proteins.
The goal of the study was to investigate the potential use of these spectroscopy and microscopy techniques to evaluate the stability of complex influenza vaccines formulations.
2. Materials and Methods 2.1 Materials
Seasonal influenza vaccines administered in the Northern Hemisphere during the 2009-2010 season were purchased from a pharmacy and stored at 4°C until use:
(a) Mutagrip® (#E7024-4) from Sanofi Pasteur, (b) Inflexal® V (#300164801) from Berna Biotech, (c) Influvac® (#S08A) from Solvay Pharma and (d) Fluarix® (#AFLUA 497AA) from GlaxoSmithKline. These inactivated trivalent vaccines produced from eggs were formulated at 15 µg per 0.5 mL of each active agent as recommended by the World Health Organisation (WHO), namely hemagglutinin protein of the strains A/Brisbane/59/2007 (H1N1)-like virus, A/Brisbane/10/2007 (H3N2)-like virus and B/Brisbane/60/2008-like virus [34, 35]. Depending on the strain, hemagglutinin protein was composed of 6-10% of aromatic amino acids. Details provided by manufacturers on vaccine type, inactivation agent and low content excipients are listed in Table 1. Prior to measurement, syringes were stored 20 min at room temperature. Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) was purchased from Invitrogen (Buchs, Switzerland). Nile Red was dissolved in ethanol to produce a 50 µM stock solution, which was filtrated with 0.22 µm filter and stored at 4°C, protected from light.
Table 1. Details on trivalent inactivated vaccines administered in the Northern Hemisphere for the 2009-2010 season.
Vaccine Product Manufacturer
Vaccine type
Inactivation agent
Buffer and other excipients per 0.5 mL dose*
(a) Mutagrip®
Sanofi Pasteur MSD
Split Triton X-100 Formaldehyde
Phosphate buffer with sodium chloride
Formaldehyde max. 0.1 mg Saccharose
Triton X-100 max. 0.5 mg Ovalbumin max. 0.05 µg Neomycin
(b) Inflexal® V Berna Biotech
Virosomal Propiolactone Potassium phosphate buffer with sodium chloride
Lecithin 117 µg (c) Influvac®
Solvay Pharma
Subunit Triton X-100 Formaldehyde
Potassium phosphate buffer with sodium chloride, potassium chloride, calcium chloride and magnesium chloride
Sucrose Formaldehyde
Cetyltrimethylammonium bromide Polysorbate 80
Gentamicin
(d) Fluarix®
GSK
Split Sodium
deoxycholate Formaldehyde
Potassium sodium phosphate buffer with sodium chloride, potassium chloride and magnesium chloride Triton X-100 max. 0.085 mg α-tocopheryl hydrogen succinate max. 0.1 mg
Ovalbumin max. 0,05 µg Polysorbate 80 max. 0.415 mg Hydrocortisone max. 0.0016 µg Gentamicin sulfate max. 0.15 µg Formaldehyde max. 50 µg Sodium deoxycholate max. 50 µg Sucrose
* The amount of excipients is indicated only if provided by manufacturers
2.2 90° light scattering
Light scattering intensity of vaccine solutions was measured between 450 and 700 nm with a Fluoromax spectrofluorometer (Spex, Stanmore, UK), equipped with a thermostated cuvette holder. Measurements were performed at 25°C in a 0.2 cm × 1 cm Hellma quartz cuvette with 120 µL of sample. Synchronous scans (λexc = λem) were recorded with a 0.01 s integration time per 1 nm increment. The excitation and emission slits were set to 1 mm. A neutral density filter, with an optical density of 2.0, was placed between the source and the sample to avoid saturation at the detector. Spectra were corrected with the water baseline. The intensity is expressed as counts per second (cps).
2.3 Intrinsic fluorescence measurements
Steady-state fluorescence emission measurements were performed at 25°C with the Fluoromax spectrofluorometer (Spex, Stanmore, UK) equipped with a thermostated cuvette holder. Fluorescence emission was monitored with a 0.01 s integration time per 1 nm increment. The fluorescence settings were optimized for each vaccine. Excitation wavelength was set to (a) 285 nm, (b) and (c) 279 nm and (d) 290 nm. The excitation and emission slits were set to (a) 1.2 mm, (b) 0.75 mm, (c) 0.6 mm and (d) 2 mm. A neutral density filter, with an optical density of 1.0, was placed between the source and the cuvette for the two split vaccines, (a) and (d), to record spectra without saturation at the detector. The intensity of fluorescence is expressed as counts per second (cps).
2.4 Fluorescence lifetime measurements
Fluorescence lifetimes were measured using time-correlated single-photon counting (TCSPC) on an IBH 5000U fluorescence lifetime spectrophotometer (Jobin Yvon Horiba, Stanmore, United Kingdom) fitted with a 279 nm excitation source and a monochromator at
the emission side. Emission wavelength was determined for each vaccine by steady-state fluorescence spectroscopy. Measurements were performed in a 0.2 cm × 1 cm Hellma quartz cuvette with 120 µL of sample. The instrument response function was measured using a dilute suspension of colloidal silica (Ludox, Aldrich, Milwaukee, USA). After reconvolution of the intensity decay with the instrument response function, the calculated data were analyzed by linear and non-linear least-squares modeling using the DAS6 software (IBH, Glasgow, United Kingdom). The mean weighted average lifetimes, τF, were calculated using the equation (1):
(1)
where τ is the fluorescence decay time and α the normalized pre-exponential factor [36].
2.5 Fluorescence microscopy with Nile Red staining
Vaccines samples were stained with Nile Red prior to observation by adding 1 µL of the 50 µM Nile Red stock solution to 50 µL of vaccine samples. The stained samples were pipetted inside Kova Glasstic slides (Hycor, Garden Grove, USA). The observations were performed on an Axiovert 200 microscope (Zeiss, Göttingen, Germany) equipped with a mercury discharge lamp and a Zeiss filter cube no. 15 (EX BP 546/12, BS FT 580, EM LP 590), using a 10x A-Plan LD objective (Zeiss, Göttingen, Germany). Images were acquired with a cooled Retiga 1300 C colour CCD camera (QImaging, Burnaby, Canada) and processed with the Openlab version 3.1.7 software (Improvision, Coventry, UK). The number of particles was averaged for 6 wells.
2.6 Characterization of vaccines stored at 4°C
Vaccines stored at 4°C and protected from light (recommended conditions by all the manufacturers), were analyzed at three time points: time 0, 4 weeks and 8 weeks. One syringe
was used per vaccine per time point. Time 0 was 5 months prior to expiration for vaccine (a) and 6 months prior to the expiration date for vaccines (b), (c) and (d) (Figure 1). For each vaccine, the most important change observed between successive time points was used as reference for the study of stresses effects.
Figure 1. 90° light scattering and intrinsic fluorescence emission spectra recorded from samples stored at 4°C of (a) Mutagrip® (Sanofi Pasteur), (b) Inflexal® V (Berna Biotech), (c) Influvac® (Solvay Pharma) and (d) Fluarix® (GSK). Samples were characterized at time 0 (solid line), 4 weeks (dashed line) and 8 weeks (dotted line) of a study started (a) 5 months before expiration and (b), (c) and (d) 6 months prior to expiration.
2.7 Effect of thermal stresses and of one freeze-thaw cycle
Seasonal influenza vaccines were stressed in their original container (syringe). One vaccine syringe was used per stress-condition. Syringes of vaccines were stored at i) 25°C for 2 weeks, ii) 25°C for 4 weeks iii) 37°C for 24 hours or iv) frozen at -24°C and kept at that temperature for 24 hours. Frozen samples were thawed within 3 minutes in a 25°C- thermostated water bath. Stressed samples were analyzed after equilibration at room temperature for 20 min. The effects of storage at 37°C for 24 hours and of one freeze-thaw cycle were obtained by comparing properties recorded for stressed samples with those measured at time 0. The effect of storages at 25°C was obtained by comparing properties recorded for stressed samples with those measured at 4 weeks. For each vaccine, the reference determined from the study of samples stored at 4°C was used to study the effect of stresses.
3. Results
3.1 Characterization of seasonal influenza vaccines stored at 4°C
Seasonal influenza vaccines stored at 4°C were characterized by 90° light scattering, intrinsic fluorescence spectroscopy, mean fluorescence lifetime and Nile Red fluorescence microscopy at three time points (time 0, 4 weeks and 8 weeks) (Figure 1). If occurring, changes in fluorescence properties may reflect physical degradation (aggregation or conformational changes) and/or chemical degradation of the protein.
At time 0, the lowest intensity of 90° light scattering, 3.6 × 106 cps at 450 nm, ILS, was measured for the subunit vaccine (c) (Figure 1 and Table 2, ILS). For split vaccines, (a) and (d), ILS of 4.0 × 106 cps and 5.3 × 106 cps were measured respectively (Figure 1 and Table 2, ILS). The highest intensity, 9.0 × 106 cps at 450 nm, was measured for the vaccine (b), based on virus like particles (Figure 1 and Table 2, ILS).
