Influence of methionine oxidation on the aggregation of recombinant human growth hormone

European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 42–52

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Research paper

Influence of methionine oxidation on the aggregation of recombinant human growth hormone Filippo Mulinacci a, Emilie Poirier b, Martinus A.H. Capelle b, Robert Gurny a, Tudor Arvinte a,b,⇑ a b

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland Therapeomic Inc., Bio Park Rosental, Mattenstrasse 22, Basel, Switzerland

a r t i c l e

i n f o

Dedicated to Hans Peter Merkle on the occasion of his 70th birthday Keywords: Aggregation Methionine oxidation Human growth hormone Stability Freeze/thawing

a b s t r a c t Oxidation of methionine (Met) residues is one of the major chemical degradations of therapeutic proteins. This chemical degradation can occur at various stages during production and storage of a biotherapeutic drug. During the oxidation process, the side chain of methionine residue undergoes a chemical modification, with the thioether group substituted by a sulfoxide group. In previous papers, we showed that oxidation of the two most accessible methionine residues of recombinant human growth hormone (r-hGH), Met14 and Met125, has no influence on the conformation of the protein [1]. However, the oxidized r-hGH is less thermally stable than the native protein [2]. In the current work, the consequences of the oxidation of these two methionine residues on the aggregation of r-hGH were investigated. The aggregation properties and kinetics of the native and oxidized r-hGH were measured in different buffers with both spectroscopic and chromatographic methods. Stabilities of oxidized and non-oxidized r-hGH were studied after storage at 37 °C and freeze/thawing cycles. Methionine oxidation influenced the aggregation properties of r-hGH. In accelerated stability studies at 37 °C, oxidized hormone aggregated more and faster than non-oxidized hormone. In freezing/thawing stability studies, it was found that oxidized r-hGH was less stable than its non-oxidized counterpart. In case of hGH, we have shown that chemical degradations such as oxidation can affect its physical stability and can induce aggregation. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Non-enzymatic degradations of proteins are in general subdivided into conformational changes (i.e. modifications of the tridimensional structure of the protein) and covalent modifications (i.e. chemical degradations of the primary structure of the protein) [3,4]. Examples of covalent modifications of proteins are oxidation, deamidation, disulfide bridges formation or scrambling, racemization, hydrolysis, glycation, and cross-linking [5,6]. These chemical degradations can involve different amino acid residues and can be influenced by pH, temperature, and ionic strength [7–9]. Among the various chemical degradations, one of the most occurring is the oxidation of methionine (Met) residues. Oxidation produces a modification of the thioether group of the methionine side chain. In the first stage of the reaction, the thioether group is substituted Abbreviations: r-hGH, recombinant human growth hormone; UV–Vis, ultraviolet–visible; 1,8-ANS, 1-anilinonaphthalene-8-sulfonic acid; RP-HPLC, reverse phase high pressure liquid chromatography; SEC, size exclusion chromatography; RT, room temperature. ⇑ Corresponding author. School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CH-1211 Geneva 4, Switzerland. Tel.: +41 76 3910208; fax: +41 615440016. E-mail address: [email protected] (T. Arvinte). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.03.015

by a sulfoxide derivative; in the second step, the sulfoxide is further oxidized to sulfone. The sulfoxide derivative is commonly observed both in vivo and in vitro; on the contrary, the sulfone derivative product is rarely observed as it requires a much stronger chemical attack [10]. Oxidation of methionine residues has been reported to influence the conformation and stability of proteins: (i) the secondary structure of parathyroid hormone (PTH) and of its biological active fragment, 1–34 PTH, are modified as a consequence of the oxidation of the methionine residues [11]; (ii) oxidation of Met388 of thrombomodulin reduces its anticoagulant cofactor activity of 90% [12]; (iii) the structure of human IgG1 Fc is modified as a consequence of methionine oxidation and the melting temperature (Tm) is significantly reduced [13]; (iv) oxidation of the Met69 of recombinant human leptin reduces to about 20% the bioactivity of the protein [14]; (v) oxidation of methionine residues in salmon calcitonin produces morphologically different aggregates [15]. However, there are a few examples where oxidation has no effect on the conformation, stability, and activity of proteins or where the effect of oxidation depends on the position of the methionine residues involved: (i) oxidation of Met14 and Met125 of human growth hormone has minimal effect on the protein activity [16,17]; (ii) oxidation of Met314 and Met315 of antithrombin does

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not affect either the thrombin-inhibitor activity or the heparin binding, while oxidation of Met17 and Met20 reduces the heparin affinity [18]. Human growth hormone is a single-chain protein consisting of 191 amino acid residues which contains three methionine residues in position Met14, Met125, and Met170 [19]. The first two methionine residues, Met14 and Met125, are partially exposed on the surface of the protein and easily oxidized by chemical treatment with hydrogen peroxide (H2O2) [20]. On the contrary, the third residue, Met170, is partially buried and not easily oxidized [21,22]. In a previous work of our group [1], the investigation of the conformation of an oxidized sample of recombinant human growth hormone (rhGH) revealed that the oxidation of the methionine residues Met14 and Met125 does not induce conformation changes in the protein. Nevertheless, the thermal stability of the oxidized r-hGH is decreased as a consequence of the oxidation [2]. In the current paper, the effect of the methionine residues oxidation on the aggregation of r-hGH is investigated. The physical stability of the oxidized rhGH is directly compared with that of the native protein in different buffers. Kinetic of the aggregation, intensity of the aggregation in accelerated stability studies at 37 °C, and the effect of freeze/ thawing are evaluated and discussed.

2. Materials and methods 2.1. Materials Hydrogen peroxide (H2O2), L-Histidine, sodium hydroxide, 1propanol, hydrochloric acid, and 7-diethylamino-3,4-benzophenoxazine-2-one (Nile Red) were supplied by Sigma–Aldrich (Sigma–Aldrich Chemie GmbH, Buchs, Switzerland). Citric acid anhydrous, Tris(hydroxymethyl)aminomethane, and L-Methionine were purchased from Fluka (Fluka GmbH, Buchs, Switzerland). Sodium phosphate mono- and di-basic and sodium chloride were acquired from Riedel-de Haën (Riedel-de Haën GmbH, Seelze, Germany). All chemicals and reagents were of analytical grade. Ultrapure water (Type I) was produced by a Millipore MilliQ Academic system (Millipore AG, Zug, Switzerland). The recombinant human growth hormone was kindly provided by Merck-Serono (Merck-Serono SA, Italy). UV transparent 96-well CostarÒ Corning (Corning Inc., New York, NY, USA) microplates were supplied by Vitaris (Vitaris SA, Baar, Switzerland). UV–Vis transparent Greiner VIEWseals™ (Greiner Bio-One GmbH, Frickenhausen, Germany) were purchased from Huber & Co. AG (Reinach, Switzerland). Quartz cuvettes for fluorescence spectroscopy were from Hellma (Hellma Schweiz AG, Zumikon, Switzerland). Slide-A-LyzerÒ dialysis cassette (Pierce Biotechnology, Thermo Fisher Scientific Inc., USA) were purchased from Perbio (Perbio Science SA, Lausanne, Switzerland). Disposable polystyrene cells were from VWR (VWR International, Nyon, Switzerland).

2.2. Preparation and chromatographic analysis of the oxidized growth hormone The oxidation was performed as previously reported [1]. The oxidized protein was characterized by reverse phase liquid chromatography (RP-HPLC) using the method described in the European Pharmacopoeia monograph for hGH [19]. The separation was performed on a Waters Alliance HT 2790 HPLC system coupled to a Waters 2487 UV–Vis detector (Waters AG, Switzerland) and a Bruker Esquire 3000+ mass spectrometer (Bruker Daltonics GmbH, Switzerland). The chromatographic peaks were identified by tryptic digestion and HPLC-MS as previously reported [1].

2.3. Intrinsic fluorescence spectroscopy The intrinsic fluorescence of the protein was measured using a SPEX Fluoromax fluorescence spectrometer (Horiba Jobin Yvon Inc., US). Fluorescence spectra were recorded at 25 °C in a quartz cuvette with a 1 cm pathlength. Settings for the measurements were the following: excitation wavelength at 280 nm, excitation slit of 0.5 mm, emission slit of 1.0 mm, and integration time of 0.1 s. The emission of the tryptophan residue Trp86 was monitored between 295 nm and 450 nm by using an optical filter (10% light transmission) to avoid saturation of the detector. The fluorescence of both the native and oxidized protein was measured in different buffers, listed in Table 1, after preparation (t = 0) and after 2 months storage at 37 °C. Solutions of oxidized and non-oxidized r-hGH in different buffers were prepared by dilution from a stock solution of r-hGH in water. The concentration of both oxidized and native hGH was 4 mg/mL in the final formulations. 2.4. Aggregation kinetics at 37 °C measured by 90° light scattering The kinetics of protein aggregation was monitored using the 90° static light scattering signal. The 90° light scattering of the samples was measured with a SPEX Fluoromax fluorescence spectrometer (Horiba Jobin Yvon Inc., US) operating in synchronous mode. In this type of experiment, the light scattering signal at a chosen wavelength is monitored over a period of time; the resulting plot shows the variations of the light scattering of the sample over time and provides an indication of the aggregation kinetics. Disposable polystyrene cells with a 1 cm pathlength were used; the cells were thermostated at 37 °C in a water bath, and the synchronous signal at 500 nm was recorded. The measurements were performed in the following order: first, the light scattering of the buffer alone was recorded for 2 min to obtain a baseline signal; then, the protein was added to the buffer solution and mixed. The light scattering signal was measured continuously. Both excitation and emission slits were 1 mm, the integration time was 1 s, and the signal was recorded every 10 s. The period of time for the analyses varied between 1 h and 10 h based on the aggregation rate of the samples. All the measurements were carried out without stirring. Protein concentration was 8 mg/mL. For those solutions which became turbid by eye during the experiment, an optical filter (10% light transmission) was used. 2.5. Size exclusion chromatography The percentages of soluble dimers and higher molecular weight species of both proteins at different pH values were measured at the beginning of the stability study (t = 0) and after 2 months of storage at 37 °C. For both oxidized and non-oxidized r-hGH, three replicates per pH value were measured. Protein concentration was about 4.5 mg/mL in all samples. The separation was achieved on a Waters Alliance HT 2790 pump with a Waters BioSuite™ UHR SEC (4.6  300 mm, 125 Å, 4 lm) (Waters AG, Switzerland) column

Table 1 List of buffers used for the stability studies. Buffers

pH

Concentration (mM)

NaCl (mM)

Sodium citrate Sodium citrate Histidine/HCl Histidine/HCl Histidine/HCl Histidine/HCl Sodium phosphate Sodium phosphate

3.0 3.0 6.5 6.5 7.5 7.5 7.5 7.5

20 20 20 20 20 20 20 20

– 100 – 100 100 – 100

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operating at room temperature with a flow rate of 0.3 mL/min. The mobile phase was the same as described in the European Pharmacopoeia monograph for hGH [19]: 63 mM phosphate buffer pH 7.0 and 1-propanol (97:3 v/v). The elution was monitored at 280 nm with a Waters 2487 UV–Vis detector (Waters AG, Switzerland), and the percentage of the different r-hGH species were given as area-under-curve (AUC) at 280 nm.

2.6. Stability at 37 °C measured by Nile Red fluorescence spectroscopy The intensity of Nile Red fluorescence in the different formulations containing oxidized and non-oxidized r-hGH was measured with a Tecan Safire2™ (Tecan Group Ltd., Männedorf, Switzerland) microplate reader. Prior to the start of the kinetic at 37 °C, Nile Red was added to the protein samples to a final concentration of 1 lM. All the samples were incubated and measured in the Safire plate reader at a temperature of 37 °C for 2 weeks. The dye was excited at 550 nm, and its fluorescence emission was recorded at 620 nm; bandwidths of 7 nm were used for both the excitation and the emission. Five measurements per sample were acquired with an integration time of 40 ls. The fluorescence emission spectra were measured using the bottom optics. Three replicates per sample were measured. The concentration of both oxidized and non-oxidized r-hGH was 4 mg/mL.

2.7. Fluorescence microscopy with Nile Red staining The presence of aggregates at the beginning of the stability study (t = 0) and after 2 months storage at 37 °C was investigated by fluorescence microscopy with the use of a staining agent. The dye used for the staining was Nile Red, at a concentration of 1 lM. Analyses were performed as described by Demeule et al. [23]. The microscope employed for the observations was an Axio Imager Z1 microscope (Zeiss, Germany) equipped with a mercury discharge lamp for fluorescence microscopy. A Zeiss filter cube no. 15 was used (EX BP 546/12, BS FT 580, EM LP 590). The pictures were acquired with an AxioCam MRm camera (Zeiss, Germany), with a 10X objective and processed using the AxioVision v4.4 (Zeiss, Germany).

3. Results and discussions 3.1. Percentage of oxidation after treatment with hydrogen peroxide As previously reported [1], the treatment with hydrogen peroxide resulted in the oxidation of more than 90% of r-hGH. The oxidation by hydrogen peroxide involved only the methionine residues in position Met14 and Met125; no oxidation of the methionine residue in position Met170 or of other amino acid residues was found. 3.2. Intrinsic fluorescence spectroscopy Human growth hormone contains eight tyrosine residues and one tryptophan residue in position Trp86; this tryptophan residue is the major contributor to the intrinsic fluorescence of the protein. In the native conformation of hGH, the tryptophan residue is partially buried inside the protein and far from the methionine residue Met14 and Met125 (Fig. 1). The fluorescence emission of Trp86 was used to investigate possible conformation changes and aggregation upon 2 months storage at 37 °C. Intrinsic fluorescence spectroscopy is a powerful tool which provides conformational and structural information of proteins. The source of the intrinsic fluorescence of protein resides in the aromatic residues tryptophan, tyrosine, and phenylalanine. Each of them has a typical fluorescence spectrum, with the position of the maximum that varies based on the residue and its electronic environment [24]. Due to their hydrophobic nature, these residues are often buried or partially buried inside the protein core. However, a conformation change can expose these residues to the surface of the protein, in closer contact with the aqueous polar environment. If this is the case, a shift of the kmax, the wavelength at which the fluorescence intensity is maximal, toward lower frequencies (red shift) is often observed. At the beginning of the stability study, the maximum of fluorescence emission was at 337 nm for both native and oxidized

2.8. Freezing/thawing stability The role of the oxidation on the freeze/thaw induced aggregation of r-hGH was investigated in different buffers. For the experiments, samples were frozen at 20 °C in a Whirlpool Easytronic freezer (Bauknecht A.G., Lenzberg, Switzerland). Before each thawing cycle, samples were stored at least 1 day at 20 °C. The effect of three successive freeze/thaw cycles was investigated by Nile Red fluorescence spectroscopy by using a Tecan Safire2™ (Tecan Group Ltd., Männedorf, Switzerland) microplate reader, Nile Red fluorescence microscopy using an Axio Imager Z1 microscope (Zeiss, Germany) and 90° light scatter using a SPEX Fluoromax spectrometer (Horiba Jobin Yvon Inc, Stanmore, UK). The following settings were used: (a) Nile Red fluorescence: excitation 550 nm and emission between 570 and 750 nm; excitation and emission slits of 7 nm both; 3 scans per sample with an integration time of 40 ls; measurements performed at RT with the bottom optics; (b) Nile Red microscopy: same settings used for the stability at 37 °C, previously described; (c) 90° light scattering: acquisition range between 450 and 750 nm, with excitation and emission slits of 1 mm both; protein concentration between 0.5 mg/mL and 8 mg/mL. All measurements were performed at room temperature. After thawing, samples were equilibrated at RT prior to analyses. Protein concentration was 4 mg/mL unless otherwise specified.

Fig. 1. Structure of human growth hormone. The three methionine residues Met14, Met125, and Met170 are marked in yellow; the tryptophan residue Trp86 is marked in blue. Image is drawn from published coordinates (PDB ID 1A22) by using PyMOL software (Delano Scientific, California, USA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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protein, regardless of the buffer used. After 2 months storage at 37 °C, the fluorescence emission maximum shifted from 337 nm to 339 nm in both oxidized and non-oxidized r-hGH. The intensity of the emitted fluorescence can provide information on the aggregation and conformation state of a protein. Fresh solutions of both oxidized and non-oxidized r-hGH in different buffers had different Trp86 fluorescence (Fig. 2). Moreover, in almost all buffers, oxidized r-hGH had lower fluorescence intensity than non-oxidized r-hGH; the only exception was in sodium citrate pH 3 in the presence of NaCl. The statistical significance of the differences between native and oxidized samples was analyzed by unpaired Student’s t-tests. In all buffers, the calculated P-values were smaller than 0.005; the only exception was sodium phosphate pH 7.5, where the difference between oxidized and native r-hGH was not significant. After 2 months at 37 °C, the fluorescence intensity of all the samples decreased (Fig. 3). Oxidized hGH samples had a more pronounced fluorescence decrease. The fluorescence intensities of the oxidized and non-oxidized protein samples, measured after 2 months at 37 °C, were also significantly different from each other, in all the tested buffers. After 2 months at 37 °C, the fluorescence intensities of both proteins were reduced. Compared to the fluorescence intensities at the beginning of the stability study, after 2 months, the biggest variations were observed in acidic conditions, especially in presence of NaCl. In sodium citrate pH 3 with NaCl, the fluorescence intensity of both native and oxidized r-hGH decreased 95%. Precipitation of the protein in the cuvette, observed in both oxidized and nonoxidized samples, may be partially responsible for this large decrease in the fluorescence intensity at pH 3 in the presence of salts. In the literature, it is reported that human growth hormone undergoes conformation changes at acidic pH [25]: the structural helical core of the hormone is conserved but becomes dynamically more flexible and surrounded by less ordered loops. These conformation changes do not affect the secondary structure, although the protein does become partially unfolded [26]. At acidic pH, some hydrophobic residues of the second and fourth loops become exposed to the surface: as a consequence, the hydrophobicity of the protein increases [25]. This increase in protein hydrophobicity could explain the poor stability of the protein in sodium citrate pH 3. The hydrophobic molecules of r-hGH tend to minimize the interactions with water and consequently aggregate and precipitate. The presence of salts, which mask and counterbalance repulsive charges of proteins, further enhances protein aggregation.

Fig. 2. Intrinsic fluorescence emission intensities at t = 0. Native r-hGH is represented in black; oxidized r-hGH in striped black. Error bars represent the standard deviation.

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Fig. 3. Intrinsic fluorescence emission intensities after 2 months storage at 37 °C. Native r-hGH is represented in black; oxidized r-hGH in striped black. Error bars represent the standard deviation.

3.3. Aggregation kinetics at 37 °C measured by 90° light scattering In our study, the aggregation kinetics of the oxidized and nonoxidized r-hGH was investigated at 37 °C by using 90° light scattering. Our results indicate that oxidized r-hGH aggregated much faster than the non-oxidized counterpart. Moreover, results also indicate that parameters such as pH, buffer type, and ionic strength of the solution affect the aggregation of oxidized and non-oxidized hormone. At pH 7.5, both in Histidine/HCl and in phosphate buffer, no increase in the 90° light scattering signal was recorded (data not shown). At this pH, neither oxidation state nor the presence of salts had an influence on the aggregation rate of the protein. At pH 6.5 in Histidine/HCl buffer, no increase in the light scattering signal of native r-hGH was observed; on the contrary, an increase was observed for the oxidized r-hGH (Fig. 4). At pH 6, the addition of salts increased the aggregation rate of the oxidized proteins and induced aggregation of the non-oxidized (data not shown). No measurement was performed between pH 4.5 and 5.5 because of the occurrence of protein precipitation in the proximity of the isoelectric point of the protein. In sodium citrate pH 3, methionine oxidation strongly destabilized the hormone. While the light scattering of the native protein remained constant, the light scattering of the oxidized sample quickly

Fig. 4. Kinetics of r-hGH aggregation in 20 mM sodium citrate pH 6.5 measured by 90° light scattering. Measurements were performed at 37 °C. Protein concentration was 8 mg/mL. Native r-hGH is represented as black line; oxidized r-hGH as gray line.

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Fig. 5. Kinetics of r-hGH aggregation in buffer pH 3 measured by 90° light scattering. Measurements were performed at 37 °C. Protein concentration was 8 mg/mL. Native r-hGH is represented as black line; oxidized r-hGH as gray line. (A) Sodium citrate 20 mM pH 3; (B) sodium citrate 20 mM pH 3 + 100 mM NaCl.

increased (Fig. 5). At pH 6, the addition of salt enhanced the rate and intensity of aggregation of both oxidized and non-oxidized rhGH. While in absence of NaCl, the light scattering of non-oxidized r-hGH remained constant, in presence of NaCl an increase in the light scattering of non-oxidized hormone was observed. The destabilizing effect of salt was even more pronounced on the oxidized hGH, where light scattering of the sample increased more than 10-folds (Fig. 5B). Protein aggregation is an important phenomenon which has consequences both in vivo and in vitro. In vivo, protein aggregation is often associated with neurodegenerative diseases such as Alzheimer’s [27,28], Parkinson’s [29,30], or prion diseases [31]; in vitro, it interferes with the production and storage of therapeutics. An understanding of the mechanisms behind this phenomenon can help to obtain more stable biotherapeutics. In solution, proteins adopt different conformations all in equilibrium with each other; in some of these possible conformations, hydrophobic residues of the proteins are more exposed to the surface. The aggregation pathway of proteins is often described to proceed through an intermediate native-like state In where the protein have partially lost of native conformations to expose some hydrophobic residues to the surface [6,32]. This intermediate In can proceed back to the native state N or further unfold to other intermediate states, as indicated in

N In In1 . . . I1 U ! A

ð1Þ

The final state is the unfolded state U. In this state, proteins tend to form aggregates [6] in order to minimize the interaction between these hydrophobic residues and water. The reaction constants for the formation of the different native-like intermediate states (In . . . I1), unfolded state (U), and aggregate (A) vary among

Fig. 6. Percentages of r-hGH monomer measured by size exclusion chromatography. (A) Results at t = 0; (B) results after 2 months storage at 37 °C. Native r-hGH is represented by black bars; oxidized r-hGH by white dotted bars. Three replicates per sample were measured; error bars represent the standard deviation of the results.

different proteins. When the rate constants for the intermediate states are much smaller than for the unfolded or aggregated state, a lag-phase for the aggregation can be observed. At pH 7.5, the oxidation of the two partially exposed methionine residue Met14 and Met125 did not have an impact on the kinetics of aggregation of r-hGH. This result can indicate that at neutral pH, both the native and oxidized r-hGH proceed through a similar aggregation pathway. In this pathway, the increase in polarity of the methionine side-chain of the oxidized r-hGH has no effect. Nevertheless, it cannot be excluded that the presence of lag-phases in both oxidized and non-oxidized r-hGH samples prevented us from observing differences in the aggregation rate of the two samples at this pH. At lower pH values, the influence of the oxidized methionine residues on r-hGH aggregation was more pronounced. The 90° light scattering signal of oxidized r-hGH increased at pH 6.5 and no lag-phase was observed; at the same pH value, the light scattering signal of non-oxidized hormone remained constant. Enhancement of methionine-oxidation-induced r-hGH aggregation was maximal at pH 3, where a higher increase in the 90° light scattering signal of oxidized r-hGH was observed (Fig. 5A). In sodium citrate pH 3, no increase in the light scattering signal of the non-oxidized hormone was recorded after 3 h. Addition of NaCl increased the light scattering signal of oxidized r-hGH by approximately 10-fold

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Fig. 8. Protein concentration in the various solutions of native and oxidized r-hGH after 2 months at 37 °C. Concentrations were measured by UV spectroscopy. Native r-hGH is represented by black bars; oxidized r-hGH by white bars. Each sample was measured in triplicate; error bars represent the standard deviation of the results.

Fig. 7. Total area of the SEC peaks of r-hGH. Protein elution was monitored at 280 nm with a UV–Vis detector. (A) Results at t = 0; (B) results after 2 months storage at 37 °C. Native r-hGH is represented by black bars; oxidized r-hGH by dotted bars. Each sample was measured in triplicate; error bars represent the standard deviation of the results.

(Fig. 5B). The presence of NaCl contributed to destabilize also the non-oxidized hormone, as shown in Fig. 5B. The destabilizing effect of acidic buffer on the human growth hormone has already been described [25,26]. Furthermore, our previous results indicated that the stability of the protein, analyzed by thermal methods, is strongly reduced in acidic buffer [2]. In our experiments, we found that the melting temperatures of the native and oxidized r-hGH at pH 3 were Tm = 69 °C and Tm = 49 °C, respectively, showing that the oxidized protein was less stable than the non-oxidized [2]. Those findings are in agreement with the aggregation kinetics results. In the presence of NaCl, the aggregation rate of both proteins is increased. These findings are also in agreement with the melting temperature values previously measured in presence of NaCl: Tm = 56 °C for the native and Tm = 27 °C for the oxidized r-hGH [2]. 3.4. Size exclusion chromatography The content of soluble r-hGH aggregates after 2 months storage at 37 °C was addressed by SEC. At time t = 0, all the samples of both native and oxidized r-hGH had a monomer content between 96% and 99% (Fig. 6A). After 2 months, small variations in the percentage of monomeric r-hGH were observed in all the samples; the only exceptions were samples of both oxidized and native r-hGH

Fig. 9. Corrected percentages of monomer r-hGH in the various protein solutions after 2 months at 37 °C. Percentages are calculated by r-hGH monomer percentages obtained by SEC analyses corrected for the area of the chromatographic peaks. Native r-hGH is represented by black bars; oxidized r-hGH by white dotted bars. Each sample was measured in triplicate; error bars represent the standard deviation of the results.

in sodium citrate pH 3 with NaCl. In this aqueous condition, both protein samples were almost completely aggregated: the average monomer contents for the native and oxidized r-hGH samples were 13% and 3%, respectively. At all other pH values, the average monomeric content was 95% (Fig. 6B). In our experiments, detection was based on the measurement of absorbance at 280 nm; area of the peaks was indicated as area under the curve at 280 nm. At the beginning of the stability study (t = 0), some difference in peak areas at 280 nm between the samples was observed (Fig. 7A); these differences were due to the nature of the protein (i.e. oxidized or non-oxidized) and to the pH value. For example, the areas of the hGH peaks in citrate pH 3 are less than half of the areas in phosphate pH 7.5. These data show that in some buffers, aggregates were already present at the beginning of the stability study since not all the injected protein was eluted from the column. After 2 months at 37 °C, decreases in the peak areas of the various solutions of oxidized and non-oxidized r-hGH were found (Fig. 7B). The biggest decreases were found in protein samples at pH 3 with NaCl, suggesting that formation of big and poorly soluble aggregates occurred. In the absence of NaCl at pH 3, the total peak area of oxidized r-hGH was less than that of the native protein.

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Fig. 10. Changes in time of Nile Red emission intensity in different solutions of native and oxidized r-hGH as function of time. In the left column, native r-hGH results are presented; in the right column, oxidized r-hGH results. From the top to the bottom, Nile Red emission intensities in sodium citrate pH 3 (A and B); Histidine/HCl pH 6.5 (C and D); Histidine/HCl pH 7.5 (E and F); sodium phosphate pH 7.5 (G and H). Full line represent Nile Red emission in absence of NaCl; broken line in presence of 100 mM NaCl. Protein concentration was 4 mg/mL and temperature was 37 °C. Each sample was measured in triplicate; error bars represent the standard deviation of the results.

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Fig. 11. Human growth hormone aggregates formed in 20 mM phosphate buffer pH 7.5 with 100 mM NaCl after 2 months at 37 °C. Aggregates are stained with 1 lM Nile Red. On the left, native r-hGH; on the right, oxidized r-hGH.

Fig. 12. Oxidized r-hGH aggregates in 20 mM buffer pH 7.5 with 100 mM NaCl stained with 1 lM Nile Red. On the left, protein in Histidine buffer; on the right, protein in phosphate buffer. Both samples were measured after 2 months at 37 °C.

These results indicate that in acidic conditions, and in the absence of salt, oxidized protein tends to aggregate more than native. Minor differences in peaks areas between oxidized and non-oxidized rhGH samples were also found at other pH values. As differences were observed in total peaks area of the various samples, protein concentration was checked by UV–Vis spectroscopy. For the measurements, a Nanodrop 1000 UV–Vis Spectrophotometer (Thermo-Scientific, Delaware, USA) was used. Protein concentration 1 was calculated taking A0:1% [33]. No differences in 280nm ¼ 0:82 cm concentrations were measured, except for protein samples at pH 3 in the presence of NaCl where reduced protein concentrations were found (Fig. 8). These findings confirm the hypothesis of the presence of poorly soluble aggregates that could not be eluted from the size exclusion column. SEC analyses did not show significant differences in the aggregation of oxidized and native r-hGH at pH 6.5 and 7.5: all samples had a monomer percentage >95%. However, the outcome of a SEC analysis is a measure of the content of soluble monomers and aggregates; the presence of insoluble or poorly soluble aggregates can lead to an underestimation of total aggregates percentages. A way to minimize this risk is by taking into account the total peaks areas. In our study, by considering the protein not eluted from the column as aggregated r-hGH, it was possible to obtain a more precise evaluation of the amount of aggregates present in all r-hGH samples after 2 months at 37 °C (Fig. 9). The total peak area of a fresh solution in water of r-hGH at 4.5 mg/mL was used as reference area. In this way, it was possible, for example, to find that after 2 months at 37 °C, oxidized and non-oxidized r-hGH in sodium phosphate pH 7.5 had a monomer content of, respectively, 68% and 81% (Fig. 9) and not 98% and 96% as previously calculated by SEC analyses (Fig. 6B).

3.5. Stability at 37 °C measured by Nile Red fluorescence spectroscopy and microscopy Nile Red is a fluorescent probe known to bind to hydrophobic surfaces [34,35]. It is widely used to analyze changes in protein conformation and aggregation [23,35–37]. In this study, physical stability of oxidized and non-oxidized r-hGH was investigated with Nile Red upon 2 months storage at 37 °C; for the study, buffers listed in Table 1 were used. In all samples, Nile Red fluorescence intensity quickly increased until it reached equilibrium after 24 h (Fig. 10). No significant differences were observed between the samples at pH 6.5 and pH 7.5: neither oxidation nor presence of salts had an effect on the intensity of Nile Red fluorescence of rhGH. However, at pH 3, some differences in the intensity of Nile Red emission were observed. At pH 3 with NaCl, Nile Red emission increased about 10-fold more than at any other pH values, both for native and oxidized r-hGH. In the absence of NaCl, the increase was much higher for the oxidized r-hGH. Data in acidic condition indicate, as reported before in this publication with other methods, that r-hGH is destabilized by acidic conditions, in particular after oxidation. In all other pH solutions, results show that oxidation has no strong effect on the Nile Red intensity emission. The intensity of Nile Red fluorescence emission increases upon binding of the dye to hydrophobic surfaces. When proteins aggregate, their native conformations are lost and hydrophobic residues may be exposed to the surface of the proteins. Nile Red binds to these residues, and its fluorescence intensity increases and the emission is shifted. Similar Nile Red fluorescence intensities in oxidized and native r-hGH samples may suggest no effect of oxidation on the unfolding and aggregation of the protein. However, even if the intensity is similar, the morphology and the size of r-hGH

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Fig. 13. Changes in Nile Red fluorescence emission intensities of different solutions of native and oxidized r-hGH as function of the number of freeze–thaw cycles. Each sample was measured in triplicate; error bars represent the standard deviation of the results.

aggregates may be different upon oxidation. In the literature, few examples of the effect of oxidation on the size and shape of protein aggregates are reported: salmon calcitonin upon oxidation forms aggregates with different morphologies [15]. To study the aggregation morphology, the samples were investigated by fluorescence microscopy after Nile Red staining, as described by Demeule et al. [23]. Fluorescence microscopy analyses revealed differences in the number and the size of protein aggregates between various r-hGH samples. All samples at pH 3, both oxidized and native, with and without NaCl, contained particles large in number and size. The high number of particles present in both samples at pH 3 made it difficult to observe differences between the native and oxidized protein (data not shown). At higher pH values, both the number and the size of the particles were reduced. At pH 6.5, oxidized rhGH samples contained more particles than native r-hGH samples; particle size was similar in both samples (data not shown). At physiological pH, oxidized protein contained more particles than native r-hGH, in particular in the presence of NaCl (Fig. 11). Analyses at pH 7.5 also showed the effect of buffer type (phosphate instead of Histidine/HCl) on the aggregation. For example, in presence of NaCl, oxidized r-hGH contained less particles in Histidine/HCl buffer than in phosphate buffer (Fig. 12). As the Nile Red fluorescence emission was similar in the two samples (Fig. 10F and H, broken lines), Nile Red microscopy results suggest that oxidized r-hGH in Histidine/HCl might contain smaller particles which are too small to be visualized. 3.6. Stability after freezing/thawing Therapeutic proteins are routinely stored as frozen solutions both during intermediate stages of production of the bulk drug substance and, often, during the long term storage of the final drug product. However, freezing and thawing of these solutions can result in denaturation and aggregation of the protein [38–40]. Protein aggregation during freezing and thawing has been attributed to several factors, such as conformation changes and partial unfolding caused by low temperature [38,41], concentration of solutes upon freezing [42], pH changes due to buffer crystallization [43,44], and exposure of the protein to ice–liquid interface. Human growth hormone is known to be susceptible to freeze-induced aggregation. Eckhardt et al. [45] reported that freezing hGH results in the formation of insoluble aggregates; they also reported that

Fig. 14. Relative increase in Nile Red fluorescence emission of different solutions of native and oxidized r-hGH after one freeze–thaw cycle. Protein solutions were frozen at 20 °C and thawed at RT. Native r-hGH is represented by black bars; oxidized are represented by black-squared bars. Each sample was measured in triplicate; error bars represent the standard deviation of the results.

pH and cooling rate have an effect on freeze-induced aggregation of the hormone. No significant increase in soluble aggregates was reported after freezing. In our study, the effect of oxidation on the freezing/thawing induced aggregation was investigated at different pHs with three orthogonal methods: Nile Red fluorescence spectroscopy, Nile Red microscopy and 90° light scattering. For both native and oxidized r-hGH, three independent samples per pH condition were prepared and analyzed. The effect of repeated freezing/thawing cycles was visible in all native and oxidized samples. After each freezing/thawing cycle, increases in Nile Red fluorescence intensities of all the samples were observed (Fig. 13). During the stability studies at 37 °C, an effect of pH on the aggregation of the protein was observed. The same effect is present on the freeze/thaw induced aggregation. As observed for the stability studies at 37 °C, acidic conditions are the most destabilizing for r-hGH, in particular after oxidation. The Nile Red fluorescence

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missing, although numerous studies have identified some causes of this phenomenon [46–50]. In this study, we showed the influence of methionine oxidation on the aggregation properties of recombinant human growth hormone. Oxidation of Met14 and Met125 enhanced aggregation of rhGH both in accelerated stability at 37 °C and in freeze/thawing studies in different formulations. The results presented here document the importance of investigating and minimizing chemical degradations early during biotherapeutical drug development.

References

Fig. 15. Aggregates of human growth hormone formed in 20 mM citrate buffer pH 3 with 100 mM NaCl after 3 freeze–thaw cycles at 20 °C. Particles are stained with 1 lM Nile Red.

increase was greater for the oxidized r-hGH samples (Fig. 14). Similar results were obtained with 90° light scattering analyses (data not shown). Staining of the protein samples with Nile Red allowed visualization of insoluble r-hGH aggregates. Differences in shape and number of particles were observed between the samples. Oxidized samples presented the larger number of particles. Furthermore, the number of particles was greater in samples containing NaCl. During freezing, solutes such as protein, buffers, salts, and excipients are concentrated; the resulting increase in protein concentration is one of the explanations for the freeze-induced aggregation of proteins. Consequently, it is possible to expect that samples with higher concentrations of r-hGH tend to aggregate more after freezing. As our results indicated that low pH and the presence of salts had a strong effect on the freeze-induced aggregation of r-hGH, influence of protein concentration was studied in sodium citrate pH 3 with NaCl. Results are shown in Fig. 15. No significant differences in the number of particles were observed between non-oxidized hGH samples at different concentrations; on the contrary, samples containing higher concentrations of oxidized r-hGH showed a larger number of particles. The differences can be explained by two mechanisms of aggregation for native and oxidized r-hGH during freezing and thawing.

4. Conclusions Aggregation of therapeutic proteins is one of the main issues during the development of biopharmaceuticals. Aggregation can occur in liquid, frozen, and lyophilized formulations. A complete understanding of the mechanisms of protein aggregation is still

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