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Stability of Human Growth Hormone in Supercritical Carbon Dioxide
Stability of Human Growth Hormone in Supercritical Carbon Dioxide CATHERINE A. KELLY,1 STEVEN M. HOWDLE,1 ANDREW NAYLOR,2 GRAHAM COXHILL,1 LAURA C. TYE,1 LISBETH ILLUM,2 ANDREW L. LEWIS2 1
School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK
2
Critical Pharmaceuticals Ltd., BioCity, Nottingham NG1 1GF, UK
Received 22 March 2011; revised 8 July 2011; accepted 12 August 2011 Published online 8 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22747 ABSTRACT: The instability of human growth hormone (hGH) to temperature and interfaces makes its formulation into injectable, sustained-release drug delivery systems challenging. A novel method of encapsulating hGH in polymeric microparticles has been developed using supercritical CO2 (scCO2 ) technology, but there is limited understanding of the stability of hGH within this system. The aim of this study was to evaluate the stability of hGH in scCO2 processing. The integrity of the protein was assessed following exposure to scCO2 using a range of different analytical techniques. Mass spectrometry showed that no peptide cleavage occurred as a result of processing or exposure to scCO2 . Size-exclusion chromatography detected formation of aggregates at high temperatures, but not as a result of the encapsulation process. Reversephase chromatography demonstrated that the production of deamidation products occurred as a function of temperature, but only at temperatures higher than those used for the encapsulation process. Circular dichroism and infrared spectroscopy demonstrated that the use of scCO2 was not detrimental to the secondary molecular structure of hGH. Together, these results show that the structural integrity of hGH is unaffected by scCO2 processing and that hGH can be successfully encapsulated in polymer microparticles using this technique. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:56–67, 2012 Keywords: supercritical fluids; human growth hormone; controlled release; stability; microencapsulation; proteins
INTRODUCTION The encapsulation of protein and peptide based drugs into sustained-release polymer microparticles is hindered by their temperature labile nature1 and ability to denature at liquid–air interfaces.2–4 Conventional methods of encapsulation, such as emulsification and spray drying, involves melting the polymer or dissolving it in a suitable organic solvent; however, these processes are generally known to denature proteins.5 A potential solution to this problem is to use an encapsulation method based on supercritical CO2 (scCO2 ), which is able to liquefy the polymers at physiological temperatures and moderate pressures.6 scCO2 is an attractive processing agent, as it is nontoxic, Correspondence to: Lisbeth Illum (Telephone: + 44-115 8820100; Fax: + 44-115-958-1565; E-mail: lisbeth.illum@illumdavis .com) Journal of Pharmaceutical Sciences, Vol. 101, 56–67 (2012) © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association
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nonflammable, relatively inexpensive and has an easily attainable critical point of 31.1◦ C and 73.8 bar. More importantly, it interacts with relatively few drugs and does not interact with peptides and proteins.7 scCO2 is soluble within polymers, entering into their free volume where it acts as a plasticiser to lower the glass transition temperature (Tg ) and the melting point (Tm ).8 This results in the polymer becoming a low viscosity liquid which can be easily mixed with proteins or other drug particles.9 We have exploited this property to develop a novel solvent free encapsulation process–CriticalMixTM (Critical Pharmaceuticals Ltd, Nottingham, UK),10 based on particles from gas saturated solutions (PGSS) process. In this process, the polymer and protein are sealed inside a high-pressure vessel. CO2 is added and the temperature and pressure are raised above the critical point to generate scCO2 , which causes the polymer to liquefy. The plasticised mixture is then stirred to create an intimate mixture
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with the solid drug particles. Finally, the polymer– drug mixture is atomised through a nozzle into a lower-pressure environment. As CO2 returns to its gaseous state, it diffuses out of the polymer droplets, causing the Tg of the polymer to rise. This solidifies the droplets, forming microparticles.11 The particles can then be collected using a cyclone. The success of this encapsulation method has been previously reported,10,12 although very little research has been published on protein stability following exposure to scCO2 .13 Proteins have been shown to be stable to other scCO2 processing techniques, for example, supercritical antisolvent and solution-enhanced dispersion.14–16 However, these techniques require solvents and are primarily used to micronise proteins and not for encapsulation. As a result, the protein experiences a very different environment as compared with the CriticalMixTM (Critical Pharmaceuticals Ltd.) process. One protein currently in development as an injectable sustained-release product is human growth hormone (hGH). In the body, hGH is produced by the pituitary gland and is responsible for a variety of physiological processes including bone and muscle development. Clinically, it is given to children with hypopituitary dwarfism, as a daily subcutaneous injection, to facilitate their growth.17 Therefore, reducing the frequency of injections would be extremely beneficial. It can also be administered to females with Turners syndrome18 and adults with HIV wasting syndrome.19 hGH exists as a single-chain peptide containing 191 amino acid residues, giving a total molecular weight of 22125 Da. The secondary structure consists of an interior of four "-helices, internally cross linked by two sulphide bonds, surrounded by remaining residues in the form of random chains and loops. This structure results in a globular protein with a hydrophobic interior.20 The most common routes of degradation observed for hGH are aggregation, oxidation and deamidation. These processes are likely to reduce the effectiveness of the protein and/or increase the possibility of immune responses within the body, but all can be quantified as discussed below.21 The secondary structure of proteins is formed by intermolecular interactions between amino acid residues. If these interactions are disrupted, by either structural changes or physical effects, protein unfolding can occur.20 There are two main techniques used to assess the secondary structure of proteins: circular dichroism (CD)22 and infrared (IR) spectroscopy.23,24 IR analysis has the additional advantage that the samples can be measured in the solid state. Many researchers have used these techniques to show that a partial loss of hGH "-helix content, coinciding with
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an increase in $-sheets, can occur as a result of solvent exposure during encapsulation by emulsion and spray-drying techniques.3,25 Aggregation is a common problem encountered with the processing of proteins, such as upon exposure to shear forces or liquid–air interfaces, they can readily unfold within the aqueous state.26 As a result of the increase in the protein size and molecular weight upon aggregation, the most effective method for their detection is size-exclusion chromatography (SEC).27 This technique has been used by many researchers to detect soluble hGH aggregate formation during emulsification, spray drying and freeze-drying.1,3,4 Oxidation of proteins can cause a partial loss in activity or, if the residue is in a critical place within the protein such as the active site, a complete loss in bioactivity.28 Oxidation can occur in the presence of hydrogen peroxide, molecular oxygen and organic solvents.29 A common technique used to determine the degree of oxidation is reverse-phase (RP) chromatography. Riggin et al.30 developed a suitable RP method capable of distinguishing the oxidised species from pure hGH, which now features within the European Pharmacopoeia.31 This and other methods have been previously used to show that oxidation occurs during both in vitro release studies from sustained-release formulations4,32 and long-term storage,33 with temperature being a significant factor.2 Deamidation in hGH can be characterised by hydrolysis of asparagine and glutamine to aspartate and glutamate, respectively.28 It occurs readily in an aqueous environment and can be influenced by both extreme pHs and high temperatures.29 Deamidation can affect the conformation of the protein, resulting in a reduction in the bioactivity.29 As the deamidated product of hGH is a charged species, RP chromatography is a useful method for its detection. Riggin et al.30 have developed a successful method for the detection of both mono- and di-deamidated forms of hGH. Many researchers2,4,32,34 have used this and similar methods to show that deamidation of hGH can occur in both solid state and in solution. The aim of the current study was to investigate the stability of hGH in scCO2 to determine whether the CriticalMixTM (Critical Pharmaceuticals Ltd.) process is a suitable processing method for encapsulation of hGH. Using a range of analytical techniques (matrix-assisted laser desorption ionization–time-offlight, SEC, RP chromatography, CD and IR), the key degradation pathways of hGH upon exposure of the dry protein to scCO2 and encapsulation into poly(D,Llactide-co-glycolide) (PLGA) microparticles were investigated.
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MATERIALS AND METHODS
Table 1.
Spray-Dried hGH Exposure Conditions
Materials Human growth hormone was purchased from Hospira (Mulgrave, Victoria, Australia) and spray dried by Upperton Ltd. (Nottingham, UK). PLGA with a lactide to glycolide ratio of 50:50 and an inherent viscosity of 0.16–0.24 dL/g (RG502H), and poly(D,L-lactide) (PLA) with an inherent viscosity of 0.16–0.24 dL/g (R202H) were obtained from Boehringer Ingelheim GmbH (Ingelheim am Rhein, R F127) was purGermany). Poloxamer 407 (Lutrol chased from BASF(Florham Park, New Jersey). Pharmaceutical grade CO2 (99.5% CO2 ) was provided by BOC (Guildford, UK) and used as received. Na2 HPO4 , NaH2 PO4 , 1-propanol and Tris base were purchased from Sigma (Gillingham, UK) and used without further modification. "-Cyano4-hydroxycinnamic acid (HCCA), anhydrous potassium bromide (KBr), dichloromethane (DCM), highperformance liquid chromatography (HPLC)-grade acetone, HPLC-grade water, sodium hydroxide, hydrochloric acid and 2-propanol were purchased from Fisher Scientific (Loughborough, UK) and, with the exception of KBr, used as received. KBr was stored in an oven set to 60◦ C for 24 h prior to use to ensure complete dryness. Spray Drying of hGH Human growth hormone was spray dried by Upperton Ltd., using a Buchi B191 spray dryer (Buchi, Oldham, UK) following the method of Maa et al.35 with minor modifications. Spray drying was performed in order to reduce the particle size of hGH and, therefore, enhance its mixing during the encapsulation procedure. Briefly, freeze-dried hGH was dissolved to a concentration of 2 mg/mL and 3.2 M ZnCl2 was added to give a Zn2+ –hGH molar ratio of 2:1. Polysorbate 20 was then added to the resulting solution to give a concentration of 0.5 mg/mL prior to spray drying. The system was equilibrated with an inlet temperature of 85◦ C, an atomisation pressure of 6.5 bar and a feed rate of 2 mL/min. The typical outlet temperature obtained was 52◦ C–55◦ C. The resulting hGH particles typically had a volume mean diameter of 2.5 :m, as measured by laser diffraction, and contained approximately 70% hGH. The particles were stored in an airtight container at–20◦ C until analysed. Exposure of Spray-Dried hGH to scCO2 Spray-dried hGH (10–15 mg) was charged into a vial and inserted into a 60-mL stainless steel highpressure vessel. The vessel was heated and pressurised with CO2 to the desired conditions (Table 1). After 1 h, the autoclave was cooled to ambient temperature and vented. An exposure time of 1 h was chosen because this is the amount of time the protein JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
Exposure Conditions Sample Not exposed to scCO2 1 2 3 4 5 6 7
Temperature (◦ C)
CO2 Pressure (Bar)
Ambient 32 32 40 45 45 60 65
Ambient 75 200 140 75 200 75 Ambient
would be exposed to scCO2 in the CriticalMixTM (Critical Pharmaceuticals Ltd.) encapsulation process. The sample was then removed and stored in a freezer at –20◦ C. Spray-dried hGH was also exposed to heat (65◦ C) for 1 h in the presence of air, as this condition was expected to aggregate and degrade the protein and could, therefore, be used as control. At least two samples were produced for each condition listed and, with the exception of MALDI-TOF mass spectrometry (MS), the results presented are the average of two batches. Encapsulation of hGH in a PLGA Matrix A high-pressure PGSS rig was used to produce PLGA microparticles with encapsulated hGH as described previously.10,36 Briefly, PLGA (1.458 g), PLA (0.162 g), Poloxamer 407 (BASF; 0.18 g) and spray-dried hGH (0.2 g) were charged into a high-pressure vessel. This vessel was heated to at least 32◦ C and pressurised with CO2 to at least 75 bar. The liquefied polymers and solid spray-dried protein particles were then stirred for 1 h, intimately mixing them together. This mixture was spayed through a nozzle, causing formation of particles which were then collected by a cyclone. Extraction of hGH from Microparticles Human growth hormone was extracted from two batches of microparticles, 24 h after processing, in order to determine its stability during the encapsulation procedure. The microparticles (10 mg) were weighed into Eppendorf microtubes (Eppendorf, Hamburg, Germany) and a 50:50 mixture of DCM and acetone (1.2 mL) was added in order to dissolve the polymer. The microtubes were centrifuged for 5 min at 7558g and then 1 mL of the supernatant was removed and discarded. This was replaced by fresh 50:50 DCM–acetone (1 mL) and the procedure was repeated three times. After the final centrifugation, the majority of the solvent was extracted and the hGH was allowed to completely dry before being analysed by the techniques described below. A control of hGH not exposed to scCO2 , but taken through the extraction procedure was compared with an untreated hGH DOI 10.1002/jps
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sample to confirm that the extraction procedure did not affect protein integrity in any way. MALDI-TOF Mass Spectrometry A Bruker Ultraflex III MALDI-TOF mass spectrometer (Bruker, Bremen, Germany) was used to identify any peptide cleavage of hGH following exposure to scCO2 and encapsulation. A small amount of each sample (Table 1) was dissolved in a 50:50 acetonitrile–water mixture (40 :L). The hGH solution (20 :L) was then added to a matrix solution (HCCA) (20 :L). This resultant mixture (0.5 :L) was spotted onto a MALDI-TOF plate and analysed in linear mode once the solvent had evaporated.
Human growth hormone was assayed and soluble aggregate formation was quantified by SEC as described in the British Pharmacopoeia27 using an Agilent 1100 series HPLC system (Agilent, Santa Clara, California). Briefly, each sample (Table 1) was dissolved in phosphate buffer (0.025 M, pH 7.0) to give a 1 mg/mL concentration, and this solution (20 :L) was injected into the system. A single mobile phase, comprising 97% phosphate buffer (0.063 M, pH 7.0) and 3% propan-2ol, was flowed through the column at 25◦ C at a flow rate of 0.6 mL/min. The column (30 cm × 7.8 mm TSKGel G2000SWXL ) (Tosoh Bioscience, Inc., San Francisco, California) was packed with a hydrophilic silica gel. The hGH peak eluted after approximately 15 min and was detected at a wavelength of 214 nm. Quantification of the percentage of monomer, dimer and oligomers present was determined by calculating the ratio of the area under the relevant peak as compared with the total area under the chromatogram. Size-exclusion chromatography was also used to determine the percentage of insoluble aggregates formed in each sample (Eq. 1):
(1)
Equation 1 gives the calculation of the percentage of insoluble aggregates by SEC: measured concentration, concentration given by SEC (mg/mL); expected concentration concentration in the original spraydried material (mg/mL). RP Chromatography Degradation of hGH via oxidation and deamidation was assessed using RP-HPLC, as described in the British Pharmacopoeia,27 with an Agilent 1100 series HPLC system (Agilent). Briefly, a butylsilyl silica gel column, 0.25 m long and 4.6 mm internal diameter (214TP54 column) (Grace-Vydac, Deerfield, Illinois), DOI 10.1002/jps
was fitted to an Agilent 1100 HPLC system (Agilent) and heated to 45◦ C. The column was washed with 50% acetonitrile–0.1% trifluoroacetic acid until the baseline was stable. The column was then equilibrated with a mobile phase of 71% 0.05 M Tris buffer–29% 1propanol at a flow rate of 1.5 mg/mL before injecting 20 :L of each sample (Table 1) containing 2 mg/mL hGH. Quantification of the percentage of native protein and mono-deamidated, di-deamidated and oxidised species present was determined by calculating the ratio of the area under the relevant peak as compared with the total area under the chromatogram.
IR Spectroscopy
Size-Exclusion Chromatography
Insoluble aggregates (%) Measured concentration × 100% =1− Expected concentration
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Infrared spectroscopy was used to detect any alterations in the secondary structure of hGH as a result of exposure to scCO2 and the encapsulation process. Discs were made by grinding hGH (2 mg) with anhydrous KBr (200 mg) using a pestle and mortar. This mixture was pressed into a disc using a Specac press (Specac, Slough, UK). The discs were analysed using a Nicolet 210 IR spectrometer (Thermo Nicolet, Madison, Wisconsin) with 1048 scans and a resolution of 4 cm−1 . Each disc was analysed twice, and two were made for each condition in order to produce representative results. These four spectra were coadded, using an OMNIC software package (Fischer Scientific UK Ltd, Loughborough, UK), to produce an average. A blank disc was also produced and its spectrum subtracted from the samples. The secondary structures of the hGH samples were determined by analysing the second derivative of the spectra between 1600 and 1700 cm−1 .25
Far-Ultraviolet CD An Applied Photophysics Pi-Star-180 spectrophotometer (Applied Photophysics Ltd., Leatherhead, UK) was used to determine whether exposure to scCO2 or the encapsulation process caused any alterations to the secondary structure of hGH. Each sample (1.5 mg) was dissolved in sodium phosphate buffer (20 mM, pH 6.8) (300 :L) and diluted to give a final concentration of 10 :mol/dm3 . Spectra were obtained from 300 :L of sample in a 1-mm path length quartz cuvette. A blank spectrum was also recorded and subtracted from the hGH spectrum. The secondary structure was investigated by recording the spectra from 250 to 200 nm in 1 nm increments at 25◦ C. The temperature was regulated using a Neslab RTE-300 circulating programmable water bath and a thermoelectric controller (Melcor, Lawrence Township, New Jersey). The mean residue ellipticity was calculated (Eq. 2) and plotted in order to remove any concentration effects. Each condition (Table 1) was analysed in JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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duplicate. [2] =
2abs 10ncl
Table 2. Mass Spectrometry Analyses of hGH Following Exposure to scCO2 , High Temperatures and the Encapsulation Process
(2)
Equation 2 gives the calculation of the mean residue ellipticity from CD: [θ], mean residue ellipticity (deg cm2 /dmol); θobs , ellipticity (mdeg); n, number of amino acid residues in the protein; c, concentration of hGH (mol/dm3 ) as determined by HPLC SEC; and l, path length (cm). Statistical Analysis Statistical analyses were performed, using Microsoft Excel, following SEC and RP chromatography. Analyses were calculated for each condition (Table 1) using analysisof variance and least significant difference at 5% confidence level. Any differences in results were considered significant if the p value was less then 0.05.
RESULTS AND DISCUSSION The secondary globular structure of hGH is built up of a hydrophobic interior of four "-helices, surrounded by the remaining residues in the form of random chains and loops. The most common changes observed for hGH as a result of instability are secondary structural rearrangement, aggregation, oxidation and deamidation.20 As these may reduce the bioactivity of the protein and/or increase the possibility of side effects (particularly immunogenicity),21 it is essential that protein integrity is maintained upon encapsulation using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process of microparticle production. Hence, the stability of hGH was assessed using a range of analytical techniques following exposure to scCO2 and/or heat and after release from the microparticles. In the scCO2 exposure studies, the hGH was in contact with scCO2 for 1 h, as this is the length of time that the protein would be exposed during batch manufacture. hGH was successfully encapsulated into polymer microparticles using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process, generating a white free-flowing powder with an hGH encapsulation efficiency of 98.3 ± 4.6%. The protein was then extracted and stability was determined. These results were compared with an unexposed control of spray-dried hGH. The aim of these studies was to identify if the CriticalMixTM (Critical Pharmaceuticals Ltd.) process is suitable for the encapsulation of hGH and to determine the appropriate processing parameters (temperature and pressure). A number of samples for each condition were analysed and, with the exception of MALDI-TOF MS, the averages are presented. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
Conditions Temperature (◦ C)
CO2 Pressure (Bar)
Not exposed to scCO2 32 75 32 200 40 140 45 75 45 200 65 In air hGH encapsulated using the PGSS rig at 40◦ C and 140 bar
m/z hGH2+
hGH+
11,074 11,067 11,067 11,065 11,064 11,069 11,069 11,072
22,150 22,125 22,118 22,119 22,112 22,128 22,125 22,115
The absence of any additional peaks and no significant reduction in the m/z values shows that peptide cleavage has not occurred at the conditions analysed.
MALDI-TOF Mass Spectrometry Matrix-assisted laser desorption ionization–time-offlight MS analyses were performed in order to detect the cleavage of the peptide backbone upon exposure to scCO2 and the encapsulation process. hGH has a molecular weight of 22125 Da,20 and if no cleavage has occurred, it should display only peaks corresponding to this mass in the spectrum. The spectrum of hGH unexposed to heat and scCO2 (Fig. 1) displays two peaks, with m/z values of 11,074.1 and 22,150.1, corresponding to hGH2+ and hGH+ ions, respectively. The presence of only hGH+ and hGH2+ peaks at approximately 22,125 and 11,067, respectively, in each of the samples (Table 2) indicates that no fragmentation has occurred as a result of exposure to scCO2 or during the encapsulation process and that the scCO2 did not react in any way with the protein. If any peptide cleavage had occurred, additional peaks or a reduction in the m/z value over 56 (corresponding to the loss of one amino acid residue) would be observed. Cleavage of hGH has not been previously reported in the literature, as it is well known that cleavage is not a standard pathway of degradation.20 Clearly, from above, there are some slight discrepancies in the m/z values between the samples. These are caused by the presence of salts and impurities within the spray-dried material, and it is within the experimental error of the system.37 hGH Aggregation Aggregation is a common problem encountered with the processing of proteins when they are exposed to shear forces or liquid–air interfaces, leading to situations in which they can readily unfold.26 SEC was used to investigate the formation of dimers and higher-order aggregates following exposure to scCO2 at different temperatures and pressures and during the polymer encapsulation process. In SEC, the hGH DOI 10.1002/jps
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Figure 1. A typical MALDI-TOF MS spectrum of hGH unexposed to heat and scCO2 . The spectrum shows two peaks at 11074.1 and 22150.1 corresponding to hGH2+ and hGH+ ions, respectively.
monomer elutes at approximately 14.4 min and the dimer at 12.7 min. The control and the samples exposed to scCO2 , heat and the encapsulation process displayed a large peak at 14.4 min, indicating that the majority of each sample remained in the monomeric form (Fig. 2). Each of the samples exposed to scCO2 and the encapsulated hGH (red trace) were found to display a small dimeric peak which is comparable in size to that of the unexposed sample (blue trace). This indicates that no further aggregation had
taken place during the exposure. However, the samples exposed to 65◦ C (green trace) showed a marked increase in the size of the dimer peak. This is consistent with previous work2,34 in which high temperatures were observed to significantly increase the rate of aggregation as a result of enhanced mobility of the hGH. Integration of the monomeric peak of each sample (Table 3) shows no significant difference among the control, the encapsulated sample and the samples
Figure 2. Size-exclusion chromatography analysis to detect the formation of aggregates on exposure to scCO2 , heat and the encapsulation process. The arrows indicate the expected retention time of the hGH dimer. Equivalent peaks were observed for the unexposed sample (blue trace) and hGH exposed to 40◦ C and 140 bar (red trace), showing that no aggregation had occurred. A larger peak was observed at 65◦ C (green trace), showing the formation of the dimer. DOI 10.1002/jps
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Table 3.
Calculation of the Percentage of Monomer Remaining Within Each Sample Conditions
Temperature
(◦ C)
CO2 Pressure (Bar)
Not exposed to scCO2 32 75 32 200 40 140 45 75 45 200 60 75 65 In air hGH encapsulated using the PGSS rig at 40◦ C and 140 bar Significant difference 5% LSD
SEC Results Monomer (%)
Dimer (%)
Oligomers (%)
99.1a
0.9a
99.0a 98.6 98.7a 98.4 97.7 97.9a 96.2 99.3a
1.0a 1.4 1.2a 1.5 1.9 1.3a 3.4 0.7a
0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.3 0.0
p = 0.009 0.71
p = 0.052 0.47
– –
Some monomeric hGH is lost on exposure to 65◦ C as a result of aggregate formation. a As a result of a limited supply of hGH, statistics could only be performed on these samples. LSD, least significant difference.
exposed to scCO2 below 45◦ C, proving that hGH is stable to aggregation at these conditions. This highlights the advantage of scCO2 processing, when encapsulating proteins into polymers for sustainedrelease drug delivery applications, over alternative encapsulating processes such as emulsification and spray drying, which can generate up to 41% aggregation of hGH.1,3,38 This is expected, as aggregation generally occurs in the presence of liquid–air and water–oil interfaces as a result of unfolding of the protein. As the CriticalMixTM (Critical Pharmaceuticals Ltd.) process encapsulates proteins as a dry powder and is carried out in a CO2 atmosphere, these interfaces do not exist and, therefore, the protein does not unfold and aggregate. Significant reductions in the monomeric content (p = 0.009), coupled with an increase in dimer and oligomers formation, were observed upon increasing the temperature further (Table 3). As discussed previously, this is as a result of the increased mobility of the protein at higher temperatures.2,34 No significant reduction in the monomeric content was observed on increasing the scCO2 pressure. Furthermore, it was shown that hGH did not undergo aggregation when encapsulated into the microparticles at 40◦ C and 140 bar. Comparison of the expected (i.e., protein content in the spray-dried powder measured prior to encapsulation or exposure to scCO2 ) and hGH concentration after encapsulation or exposure to scCO2 permits assessment of the formation of insoluble aggregates that would be unavoidably excluded from the SEC analysis. Insoluble aggregates are thought to form, initially, as soluble aggregates which then act as nucleation sites for further aggregation until the resulting aggregate becomes insoluble.39 Figure 3 shows the results for hGH exposed to 75 bar scCO2 at various temperatures. No significant difference (p = 0.132) was observed in insoluble aggregation formation between JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
the four temperatures and the control. This indicates that in the solid state, the spray-dried hGH was surprisingly stable to the temperatures tested over the 1-h exposure period. Although not significantly different, an increase in the percentage of the insoluble aggregates was observed at 90◦ C. This is consistent with the increase in dimer formation observed on raising the temperature, highlighting that aggregation is temperature dependent. The formation of insoluble aggregates is important, as it would reduce the bioavailability of the protein and could, therefore, have a negative impact upon efficacy. Secondly, the insoluble aggregate could be more immunogenic and may increase the risk of patients generating neutralising antibodies to hGH or other side effects.
hGH Oxidation and Deamidation Oxidation of proteins can cause a partial or complete loss in the bioactivity,28 whereas deamidation can affect the conformation of the protein,29 and it occurs either in an aqueous environment or in the solid state as a result of the presence of moisture. RP chromatography was used to investigate the oxidation and deamidation of hGH following exposure to scCO2 and/or heat and encapsulation into polymeric microparticles. Integration of the main hGH peak gives the percentage of remaining native hGH. Close examination of the results obtained for various conditions (Table 4) reveals no significant change on altering the scCO2 pressure, but a significant reduction (p = 0.047) in the native hGH content upon increasing the temperature. It is well established that the extent of deamidation of hGH is temperature dependent.2,34 Furthermore, hGH encapsulated using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process at 40◦ C and 140 bar did not undergo oxidation or deamidation (Table 4). DOI 10.1002/jps
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Figure 3. Assessment of insoluble aggregate formation of hGH exposed to CO2 at 75 bar and various temperatures. No significant difference was detected among the four temperatures (p = 0.132).
Further examination of the chromatograms used to calculate these results provides information on the source of this degradation (Fig. 4 and Table 4). In a degraded sample, the hGH native peak is eluted after approximately 33.5 min, and the mono-deamidated, di-deamidated and oxidised peaks appear prior to it at 27.5, 23 and 25 min, respectively.30 The control (blue trace) and hGH exposed to 40◦ C at 140 bar (red trace) show the presence of a small peak at approximately 27 min, which becomes more pronounced at 65◦ C (green trace), indicating that hGH degrades principally by deamidation. Integration of these peaks
(Table 4), although not significant, shows a general trend of increasing mono-deamidation on raising the temperature, with the change in pressure appearing to have little effect. This mirrors the earlier work which reported that hGH can deamidate when stored at high temperatures.34 A peak at 23 min also becomes distinctive at 65◦ C, indicating that hGH undergoes both mono- and di-deamidation at high temperatures. Previous work has showed asparagine-149 to be the principal site for deamidation, with a slower reaction also occurring at asparagine-152.40,41 This was attributed to these residues being situated within
Table 4. Calculation of the Percentage of Native Protein, Mono-Deamidated, Di-Deamidated and Oxidised Species Present after Processing Conditions Temperature (◦ C)
CO2 Pressure (Bar)
Not exposed to scCO2 32 75 32 200 40 140 45 75 45 200 60 75 65 In air hGH encapsulated using the PGSS rig at 40◦ C and 140 bar Significant difference 5% LSD
RP Results Native hGH (%)
Mono-Deamidated (%)
Di-Deamidated (%)
Oxidated (%)
97.4a
1.9a
0.1a
97.8a 96.3 96.9a 95.9 95.1 96.2a 92.8 98.2a
1.6a 2.4 2.4a 2.4 3.5 2.7a 4.8 1.1a
0.1a 0.8 0.1a 0.7 0.6 0.4a 1.0 0.0a
0.5a 0.4a 0.4 0.5a 0.9 0.3 0.5a 1.3 0.4a
p = 0.047 1.33
p = 0.073 1.19
p = 0.33 0.33
p = 0.84 0.34
A significant loss is observed upon increasing the temperature, although this is not reciprocated in the encapsulated hGH. A much greater loss (4%) occurs upon exposure to 65◦ C. a As a result of a limited supply of hGH, statistics could only be performed on these samples. LSD, least significant difference.
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Figure 4. RP analysis to detect the presence of degradation products upon exposure to scCO2 and/or heat. The ↓, and ∗ represent the expected retention times of mono-deamidated, dideamidated and oxidised products, respectively. An increase in the intensity of the deamidation peaks was observed at 65◦ C, showing that high temperatures are capable of degrading hGH.
a flexible, exposed loop of the protein, making them easily accessible. It is likely, therefore, that at low temperatures, only asparagine-149 deamidates, but upon increasing the temperature, asparagine-152 also becomes susceptible.33,41
Oxidation was not observed when in the presence of CO2 . This is expected, as either hydrogen peroxide, molecular oxygen or organic solvents must be present for the amino acid residues to be oxidised.29 This analysis highlights another key advantage of scCO2
Figure 5. CD analysis following exposure to scCO2 . The similarity in the shape of each of the exposed samples to the control sample demonstrates that no change has occurred in the secondary structure. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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processing over conventional encapsulation methods, as organic solvents are not required and, therefore, degradation is reduced. Far-Ultraviolet CD Assessment of the conformation and stability of hGH was performed by measuring the CD properties of spray-dried hGH and hGH exposed to heat, scCO2 and the encapsulation process. CD has been previously used to probe the secondary structure of hGH in solution, resulting in two strong minima at 209 and 221 nm, indicative of an "-helical structure.20 The CD results obtained in the present study (Fig. 5) are consistent with this theory, confirming the secondary structure of hGH to be "-helical. Each of the samples analysed, including hGH exposed to high temperatures, showed similar "-helical traces, demonstrating that scCO2 and/or heat and the encapsulation process do not alter the secondary structure of hGH. This was expected, as previous work has shown the secondary structure of hGH to be stable in the solid state to temperatures in excess of 150◦ C.42 Each sample displays different absorbances; however, as the ratio between the two troughs (with the exception of the extracted sample) remains consistent, this is not indicative of a change in the secondary structure. As small concentrations are required for CD as compared with SEC, these differences in absorption are likely to be generated by the experimental error during the dilution from bulk SEC samples. A slight deviation was observed in the magnitude of
65
the peak at 221 nm for the extracted sample. This increase would suggest a greater "-helix structure; however, the change is only slight and, therefore, this is not conclusive. Many researchers have shown that a partial loss of hGH "-helix content, coinciding with an increase in the $-sheets, can occur as a result of solvent exposure during encapsulation by emulsion and spraydrying techniques as a result of exposure to liquid–air interfaces.3,25 Therefore, these data again highlight a significant advantage of using scCO2 processing to encapsulate proteins for controlled-release drug delivery, as solvents are not required and, therefore, no liquid–air or oil–water interfaces are present. IR Spectrometry The second derivative of an IR spectrum provides information about the secondary structure of proteins in the solid state,1,43 and has the additional advantage that samples can be measured in the solid state. These data can be used to detect any structural rearrangements caused by exposure to scCO2 and the encapsulation process. The second derivative of the IR spectrum of hGH (Fig. 6) shows four discrete peaks at approximately 1640, 1654, 1683 and 1695 cm−1 . These are indicative of extended loop structures, an "-helix and a $-turn, respectively.44 The intensities of these peaks provide qualitative information about the ratios of these structures within hGH. The similarity of these four peaks among the unexposed, scCO2 and/or heat exposed and extracted sample shows that
Figure 6. The second derivative of the IR spectrum between 1600 and 1700 cm−1 , illustrating that no alteration occurs in the secondary structure of hGH as a result of exposure to scCO2 and the encapsulation process. DOI 10.1002/jps
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no alterations have occurred in the secondary structure. Each of the conditions analysed displayed similar traces. These results are consistent with those obtained by CD, proving that the secondary structure of hGH is stable to scCO2 , heat and the encapsulation process.
CONCLUSION This work has shown that hGH can be successfully encapsulated within polymers, using an encapsulation method based on exposure of polymers to scCO2 , without degrading or denaturing the hGH. MALDI-TOF MS analyses demonstrated that no peptide cleavage occurred as a result of exposure to scCO2 or during the encapsulation procedure, and that the scCO2 did not react with the protein. CD and IR analyses concluded that the secondary structure remained unaltered during CO2 exposure and encapsulation. SEC and RP demonstrated that both aggregation and deamidation were influenced by temperature but not pressure. SEC showed the occurrence of aggregation at 65◦ C resulting in a loss in the hGH monomer content of 2.5%. No significant aggregation was observed following exposure to scCO2 below 45◦ C or the encapsulation process. RP chromatography revealed that the extent of deamidation increased upon raising the temperature. Exposure to 65◦ C showed the occurrence of both mono- and di-deamidation, illustrating that both asparagine 149 and 152 are deamidated. Deamidation was not observed following the encapsulation procedure, ascertaining that the polymers and excipients present in the spray-dried material are able to protect the protein. Comparison of these results to similar studies following other encapsulation methods has highlighted the enhanced stability when processing using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process to encapsulate proteins in polymers for injectable sustained-release drug delivery applications.
ACKNOWLEDGMENTS This work was funded by the Engineering and Physical Sciences Research Council and Critical Pharmaceuticals Ltd. The authors also gratefully acknowledge Professor Mark Searle for his expertise and advice with CD. Thanks also to Mr Peter Fields, Mr Richard Wilson and Mr Paul Gaetto of University of Nottingham technical workshop.
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2. Pikal MJ, Dellerman KM, Roy ML, Riggin RM. 1991. The effects of formulation variables on the stability of freeze-dried human growth hormone. Pharm Res 8(4):427–436. 3. Jalalipour M, Gilani K, Tajerzadeh H, Najafabadi AR, Barghi M. 2008. Characterization and aerodynamic evaluation of spray dried recombinant human growth hormone using protein stabilizing agents. Int J Pharm 352(1–2):209–216. 4. Kim HK, Park TG. 1999. Microencapsulation of human growth hormone within biodegradable polyester microspheres: Protein aggregation stability and incomplete release mechanism. Biotechnol Bioeng 65(6):659–667. 5. Brandau DT, Jones LS, Wiethoff CM, Rexroad J, Middaugh CR. 2003. Thermal stability of vaccines. J Pharm Sci 92(2):218–231. 6. Howdle SM, Watson MS, Whitaker MJ, Popov VK, Davies MC, Mandel FS, Wang JD, Shakesheff KM. 2001. Supercritical fluid mixing: Preparation of thermally sensitive polymer composites containing bioactive materials. Chem Commun (01):109–110. 7. Davies OR, Lewis AL, Whitaker MJ, Tai HY, Shakesheff KM, Howdle SM. 2008. Applications of supercritical CO2 in the fabrication of polymer systems for drug delivery and tissue engineering. Adv Drug Deliv Rev 60(3):373–387. 8. Shieh YT, Su JH, Manivannan G, Lee PHC, Sawan SP, Spall WD. 1996. Interaction of supercritical carbon dioxide with polymers.2. Amorphous polymers. J Appl Polym Sci 59(4):707–717. 9. Cooper AI. 2000. Polymer synthesis and processing using supercritical carbon dioxide. J Mater Chem 10(2):207–234. 10. Jordan F, Naylor A, Kelly CA, Howdle SM, Lewis A, Illum L. 2010. Sustained release hGH microsphere formulation produced by a novel supercritical fluid technology: In vivo studies. J Control Release 141(2):153–160. 11. Woods HM, Silva MMCG, Nouvel C, Shakesheff KM, Howdle SM. 2004. Materials processing in supercritical carbon dioxide: Surfactants, polymers and biomaterials. J Mater Chem 14:1663–1678. 12. Hao JY, Whitaker MJ, Wong B, Serhatkulu G, Shakesheff KM, Howdle SM. 2004. Plasticization and spraying of poly (DL-lactic acid) using supercritical carbon dioxide: Control of particle size. J Pharm Sci 93(4):1083–1090. 13. Velega SP, Carlfors J. 2005. Supercritical fluids processing of recombinant human growth hormone. Drug Dev Ind Pharm 31(2):135–149. 14. Nesta DP, Elliott JS, Warr JP. 2000. Supercritical fluid precipitation of recombinant human immunoglobulin from aqueous solutions. Biotechnol Bioeng 67(4):457–464. 15. Mollmann SH, Bukrinsky JT, Elofsson U, Elversson J, Frokjaer S, Thalberg K, Millqvist-Fureby A. 2006. The stability of insulin in solid formulations containing melezitose and starch. Effects of processing and excipients. Drug Dev Ind Pharm 32(6):765–778. 16. Yeo SD, Lim GB, Debenedetti PG, Bernstein H. 1993. Formation of microparticulate protein powders using a supercritical fluid antisolvent. Biotechnol Bioeng 41(3):341–346. 17. Tanner JM, Whitehouse RH. 1967. Growth response of 26 children with short stature given human growth hormone. Br Med J 2(5544):69–75. 18. Carel JC, Mathivon L, Gendrel C, Ducret JP, Chaussain JL. 1998. Near normalization of final height with adapted doses of growth hormone in Turner’s syndrome. J Clin Endocrinol Metab 83(5):1462–1466. 19. Schambelan M, Mulligan K, Grunfeld C, Daar ES, LaMarca A, Kotler DP, Wang J, Bozzette SA, Breitmayer JB, Hurley A, Javaly K, Pitt J, Pang M, Heald AE, Shipp KW, Pearce D, Sova K, Pate P, Boswell SL, Flynn T, Polsky B, Haverstick L, Rothkopf MM, Bliffen DL, Rosenfeld R, Pierson RN, Winson G, Tai V, Bennison WM, Kennedy SM, Neu TK, DOI 10.1002/jps
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Robinson CE, Schwalbert RJ, LaMarca D, Kuritzkes DR, Ray MG, Mellors JW, Balser BE, Balser JP. 1996. Recombinant human growth hormone in patients with HIV-associated wasting. A randomized, placebo-controlled trial. Ann Intern Med 125(11):873–882. Pearlman R, Bewley TA. 1993. Stability and characterization of human growth hormone. In Stability and characterization of protein and peptide drugs: case histories; Wang YJ, Pearlman R, Eds. New York: Plenum Press, pp 1–58. Wright J. 2008. Overview of protein formulation and delivery. In Protein formulation and delivery; McNally EJ, Hastedt JE, Eds. 2nd ed. New York: Informa Healthcare, pp 1–6. Greenfield NJ. 1996. Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal Biochem 235(1):1–10. Pelton JT, McLean LR. 2000. Spectroscopic methods for analysis of protein secondary structure. Anal Biochem 277(2):167–176. Surewicz WK, Mantsch HH, Chapman D. 1993. Determination of protein secondary structure by Fourier transform infrared spectroscopy: A critical assessment. Biochemistry 32(2):389–394. Yang TH, Dong A, Meyer J, Johnson OL, Cleland JL, Carpenter JF. 1999. Use of infrared spectroscopy to assess secondary structure of human growth hormone with biodegradable microspheres. J Pharm Sci 88(2):161–165. Hoffmann H, Pisch-Heberle S. 2008. Analytical methods and stability testing of biopharmaceuticals. In Protein formulation and delivery; McNally EJ, Hastedt JE, Eds. 2nd ed. New York: Informa Healthcare, pp 73–109. British Pharmacopoeia. 2009. London: The Stationary Office. Bummer PM. 2008. Chemical considerations in protein and peptide stability. In Protein formulation and delivery; McNally EJ, Hastedt JE, Eds. 2nd ed. New York: Informa Healthcare, pp 7–42. Banga AK. 2006. Therapeutic peptides and proteins: Formulation, processing and delivery. Florida: CRC Press. Riggin RM, Dorulla GK, Miner DJ. 1987. A reversed-phase high-performance liquid chromatographic method for characterization of biosynthetic human growth hormone. Anal Biochem 167(1):199–209. European Pharmacopoeia 2007, 6th ed. 1997. Council of Europe. Cleland JL, Mac A, Boyd B, Yang J, Duenas ET, Yeung D, Brooks D, Hsu C, Chu H, Mukku V, Jones AJS. 1997. The stability of recombinant human growth hormone in poly(lactic-co-glycolic acid) (PLGA) microspheres. Pharm Res 14(4):420–425.
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33. Catai JR, Torano JS, Jongen P, de Jong GJ, Somsen GW. 2007. Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection. J Chromatogr B 852(1–2):160–166. 34. Salnikova MS, Middaugh CR, Rytting JH. 2008. Stability of lyophilized human growth hormone. Int J Pharm 358(1–2):108–113. 35. Maa YF, Nguyen PAT, Hsu SW. 1998. Spray-drying of air–liquid interface sensitive recombinant human growth hormone. J Pharm Sci 87(2):152–159. 36. Hao JY, Whitaker MJ, Serhatkulu G, Shakesheff KM, Howdle SM. 2005. Supercritical fluid assisted melting of poly(ethylene glycol): A new solvent-free route to microparticles. J Mater Chem 15(11):1148–1153. 37. Bird GH, Shin JA. 2002. MALDI-TOF mass spectrometry characterization of recombinant hydrophobic mutants containing the GCN4 basic region/leucine zipper motif. Biochim Biophys Acta 1597(2):252–259. 38. Jiang WL, Schwendeman SP. 2001. Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends. Pharm Res 18(6):878–885. 39. Laurence JS, Middaugh CR. 2010. Fundamental structures and behaviors of proteins. In Aggregation of therapeutic proteins, Wang W, Roberts CJ, Eds. Hoboken: John Wiley & Sons, Inc. 40. Karlsson G, Gellerfors P, Persson A, Noren B, Edlund PO, Sandberg C, Birnbaum S. 1999. Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography. J Chromatogr A 855(1):147–155. 41. Becker GW, Tackitt PM, Bromer WW, Lefeber DS, Riggin RM. 1988. Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone. Biotech Appl Biochem 10(4):326–337. 42. Pikal MJ, Rigsbee D, Roy ML. 2008. Solid state stability of proteins III: Calorimetric (DSC) and spectroscopic (FTIR) characterization of thermal denaturation in freeze dried human growth hormone (hGH). J Pharm Sci 97(12):5122– 5131. 43. Carrasquillo KG, Costantino HR, Cordero RA, Hsu CC, Griebenow K. 1999. On the structural preservation of recombinant human growth hormone in a dried film of a synthetic biodegradable polymer. J Pharm Sci 88(2):166– 173. 44. Holloway PW, Mantsch HH. 1989. Structure of cytochromeB5 in solution by Fourier-transform infrared spectroscopy. Biochemistry 28(3):931–935.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK
2
Critical Pharmaceuticals Ltd., BioCity, Nottingham NG1 1GF, UK
Received 22 March 2011; revised 8 July 2011; accepted 12 August 2011 Published online 8 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22747 ABSTRACT: The instability of human growth hormone (hGH) to temperature and interfaces makes its formulation into injectable, sustained-release drug delivery systems challenging. A novel method of encapsulating hGH in polymeric microparticles has been developed using supercritical CO2 (scCO2 ) technology, but there is limited understanding of the stability of hGH within this system. The aim of this study was to evaluate the stability of hGH in scCO2 processing. The integrity of the protein was assessed following exposure to scCO2 using a range of different analytical techniques. Mass spectrometry showed that no peptide cleavage occurred as a result of processing or exposure to scCO2 . Size-exclusion chromatography detected formation of aggregates at high temperatures, but not as a result of the encapsulation process. Reversephase chromatography demonstrated that the production of deamidation products occurred as a function of temperature, but only at temperatures higher than those used for the encapsulation process. Circular dichroism and infrared spectroscopy demonstrated that the use of scCO2 was not detrimental to the secondary molecular structure of hGH. Together, these results show that the structural integrity of hGH is unaffected by scCO2 processing and that hGH can be successfully encapsulated in polymer microparticles using this technique. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:56–67, 2012 Keywords: supercritical fluids; human growth hormone; controlled release; stability; microencapsulation; proteins
INTRODUCTION The encapsulation of protein and peptide based drugs into sustained-release polymer microparticles is hindered by their temperature labile nature1 and ability to denature at liquid–air interfaces.2–4 Conventional methods of encapsulation, such as emulsification and spray drying, involves melting the polymer or dissolving it in a suitable organic solvent; however, these processes are generally known to denature proteins.5 A potential solution to this problem is to use an encapsulation method based on supercritical CO2 (scCO2 ), which is able to liquefy the polymers at physiological temperatures and moderate pressures.6 scCO2 is an attractive processing agent, as it is nontoxic, Correspondence to: Lisbeth Illum (Telephone: + 44-115 8820100; Fax: + 44-115-958-1565; E-mail: lisbeth.illum@illumdavis .com) Journal of Pharmaceutical Sciences, Vol. 101, 56–67 (2012) © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association
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nonflammable, relatively inexpensive and has an easily attainable critical point of 31.1◦ C and 73.8 bar. More importantly, it interacts with relatively few drugs and does not interact with peptides and proteins.7 scCO2 is soluble within polymers, entering into their free volume where it acts as a plasticiser to lower the glass transition temperature (Tg ) and the melting point (Tm ).8 This results in the polymer becoming a low viscosity liquid which can be easily mixed with proteins or other drug particles.9 We have exploited this property to develop a novel solvent free encapsulation process–CriticalMixTM (Critical Pharmaceuticals Ltd, Nottingham, UK),10 based on particles from gas saturated solutions (PGSS) process. In this process, the polymer and protein are sealed inside a high-pressure vessel. CO2 is added and the temperature and pressure are raised above the critical point to generate scCO2 , which causes the polymer to liquefy. The plasticised mixture is then stirred to create an intimate mixture
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with the solid drug particles. Finally, the polymer– drug mixture is atomised through a nozzle into a lower-pressure environment. As CO2 returns to its gaseous state, it diffuses out of the polymer droplets, causing the Tg of the polymer to rise. This solidifies the droplets, forming microparticles.11 The particles can then be collected using a cyclone. The success of this encapsulation method has been previously reported,10,12 although very little research has been published on protein stability following exposure to scCO2 .13 Proteins have been shown to be stable to other scCO2 processing techniques, for example, supercritical antisolvent and solution-enhanced dispersion.14–16 However, these techniques require solvents and are primarily used to micronise proteins and not for encapsulation. As a result, the protein experiences a very different environment as compared with the CriticalMixTM (Critical Pharmaceuticals Ltd.) process. One protein currently in development as an injectable sustained-release product is human growth hormone (hGH). In the body, hGH is produced by the pituitary gland and is responsible for a variety of physiological processes including bone and muscle development. Clinically, it is given to children with hypopituitary dwarfism, as a daily subcutaneous injection, to facilitate their growth.17 Therefore, reducing the frequency of injections would be extremely beneficial. It can also be administered to females with Turners syndrome18 and adults with HIV wasting syndrome.19 hGH exists as a single-chain peptide containing 191 amino acid residues, giving a total molecular weight of 22125 Da. The secondary structure consists of an interior of four "-helices, internally cross linked by two sulphide bonds, surrounded by remaining residues in the form of random chains and loops. This structure results in a globular protein with a hydrophobic interior.20 The most common routes of degradation observed for hGH are aggregation, oxidation and deamidation. These processes are likely to reduce the effectiveness of the protein and/or increase the possibility of immune responses within the body, but all can be quantified as discussed below.21 The secondary structure of proteins is formed by intermolecular interactions between amino acid residues. If these interactions are disrupted, by either structural changes or physical effects, protein unfolding can occur.20 There are two main techniques used to assess the secondary structure of proteins: circular dichroism (CD)22 and infrared (IR) spectroscopy.23,24 IR analysis has the additional advantage that the samples can be measured in the solid state. Many researchers have used these techniques to show that a partial loss of hGH "-helix content, coinciding with
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an increase in $-sheets, can occur as a result of solvent exposure during encapsulation by emulsion and spray-drying techniques.3,25 Aggregation is a common problem encountered with the processing of proteins, such as upon exposure to shear forces or liquid–air interfaces, they can readily unfold within the aqueous state.26 As a result of the increase in the protein size and molecular weight upon aggregation, the most effective method for their detection is size-exclusion chromatography (SEC).27 This technique has been used by many researchers to detect soluble hGH aggregate formation during emulsification, spray drying and freeze-drying.1,3,4 Oxidation of proteins can cause a partial loss in activity or, if the residue is in a critical place within the protein such as the active site, a complete loss in bioactivity.28 Oxidation can occur in the presence of hydrogen peroxide, molecular oxygen and organic solvents.29 A common technique used to determine the degree of oxidation is reverse-phase (RP) chromatography. Riggin et al.30 developed a suitable RP method capable of distinguishing the oxidised species from pure hGH, which now features within the European Pharmacopoeia.31 This and other methods have been previously used to show that oxidation occurs during both in vitro release studies from sustained-release formulations4,32 and long-term storage,33 with temperature being a significant factor.2 Deamidation in hGH can be characterised by hydrolysis of asparagine and glutamine to aspartate and glutamate, respectively.28 It occurs readily in an aqueous environment and can be influenced by both extreme pHs and high temperatures.29 Deamidation can affect the conformation of the protein, resulting in a reduction in the bioactivity.29 As the deamidated product of hGH is a charged species, RP chromatography is a useful method for its detection. Riggin et al.30 have developed a successful method for the detection of both mono- and di-deamidated forms of hGH. Many researchers2,4,32,34 have used this and similar methods to show that deamidation of hGH can occur in both solid state and in solution. The aim of the current study was to investigate the stability of hGH in scCO2 to determine whether the CriticalMixTM (Critical Pharmaceuticals Ltd.) process is a suitable processing method for encapsulation of hGH. Using a range of analytical techniques (matrix-assisted laser desorption ionization–time-offlight, SEC, RP chromatography, CD and IR), the key degradation pathways of hGH upon exposure of the dry protein to scCO2 and encapsulation into poly(D,Llactide-co-glycolide) (PLGA) microparticles were investigated.
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Table 1.
Spray-Dried hGH Exposure Conditions
Materials Human growth hormone was purchased from Hospira (Mulgrave, Victoria, Australia) and spray dried by Upperton Ltd. (Nottingham, UK). PLGA with a lactide to glycolide ratio of 50:50 and an inherent viscosity of 0.16–0.24 dL/g (RG502H), and poly(D,L-lactide) (PLA) with an inherent viscosity of 0.16–0.24 dL/g (R202H) were obtained from Boehringer Ingelheim GmbH (Ingelheim am Rhein, R F127) was purGermany). Poloxamer 407 (Lutrol chased from BASF(Florham Park, New Jersey). Pharmaceutical grade CO2 (99.5% CO2 ) was provided by BOC (Guildford, UK) and used as received. Na2 HPO4 , NaH2 PO4 , 1-propanol and Tris base were purchased from Sigma (Gillingham, UK) and used without further modification. "-Cyano4-hydroxycinnamic acid (HCCA), anhydrous potassium bromide (KBr), dichloromethane (DCM), highperformance liquid chromatography (HPLC)-grade acetone, HPLC-grade water, sodium hydroxide, hydrochloric acid and 2-propanol were purchased from Fisher Scientific (Loughborough, UK) and, with the exception of KBr, used as received. KBr was stored in an oven set to 60◦ C for 24 h prior to use to ensure complete dryness. Spray Drying of hGH Human growth hormone was spray dried by Upperton Ltd., using a Buchi B191 spray dryer (Buchi, Oldham, UK) following the method of Maa et al.35 with minor modifications. Spray drying was performed in order to reduce the particle size of hGH and, therefore, enhance its mixing during the encapsulation procedure. Briefly, freeze-dried hGH was dissolved to a concentration of 2 mg/mL and 3.2 M ZnCl2 was added to give a Zn2+ –hGH molar ratio of 2:1. Polysorbate 20 was then added to the resulting solution to give a concentration of 0.5 mg/mL prior to spray drying. The system was equilibrated with an inlet temperature of 85◦ C, an atomisation pressure of 6.5 bar and a feed rate of 2 mL/min. The typical outlet temperature obtained was 52◦ C–55◦ C. The resulting hGH particles typically had a volume mean diameter of 2.5 :m, as measured by laser diffraction, and contained approximately 70% hGH. The particles were stored in an airtight container at–20◦ C until analysed. Exposure of Spray-Dried hGH to scCO2 Spray-dried hGH (10–15 mg) was charged into a vial and inserted into a 60-mL stainless steel highpressure vessel. The vessel was heated and pressurised with CO2 to the desired conditions (Table 1). After 1 h, the autoclave was cooled to ambient temperature and vented. An exposure time of 1 h was chosen because this is the amount of time the protein JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
Exposure Conditions Sample Not exposed to scCO2 1 2 3 4 5 6 7
Temperature (◦ C)
CO2 Pressure (Bar)
Ambient 32 32 40 45 45 60 65
Ambient 75 200 140 75 200 75 Ambient
would be exposed to scCO2 in the CriticalMixTM (Critical Pharmaceuticals Ltd.) encapsulation process. The sample was then removed and stored in a freezer at –20◦ C. Spray-dried hGH was also exposed to heat (65◦ C) for 1 h in the presence of air, as this condition was expected to aggregate and degrade the protein and could, therefore, be used as control. At least two samples were produced for each condition listed and, with the exception of MALDI-TOF mass spectrometry (MS), the results presented are the average of two batches. Encapsulation of hGH in a PLGA Matrix A high-pressure PGSS rig was used to produce PLGA microparticles with encapsulated hGH as described previously.10,36 Briefly, PLGA (1.458 g), PLA (0.162 g), Poloxamer 407 (BASF; 0.18 g) and spray-dried hGH (0.2 g) were charged into a high-pressure vessel. This vessel was heated to at least 32◦ C and pressurised with CO2 to at least 75 bar. The liquefied polymers and solid spray-dried protein particles were then stirred for 1 h, intimately mixing them together. This mixture was spayed through a nozzle, causing formation of particles which were then collected by a cyclone. Extraction of hGH from Microparticles Human growth hormone was extracted from two batches of microparticles, 24 h after processing, in order to determine its stability during the encapsulation procedure. The microparticles (10 mg) were weighed into Eppendorf microtubes (Eppendorf, Hamburg, Germany) and a 50:50 mixture of DCM and acetone (1.2 mL) was added in order to dissolve the polymer. The microtubes were centrifuged for 5 min at 7558g and then 1 mL of the supernatant was removed and discarded. This was replaced by fresh 50:50 DCM–acetone (1 mL) and the procedure was repeated three times. After the final centrifugation, the majority of the solvent was extracted and the hGH was allowed to completely dry before being analysed by the techniques described below. A control of hGH not exposed to scCO2 , but taken through the extraction procedure was compared with an untreated hGH DOI 10.1002/jps
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sample to confirm that the extraction procedure did not affect protein integrity in any way. MALDI-TOF Mass Spectrometry A Bruker Ultraflex III MALDI-TOF mass spectrometer (Bruker, Bremen, Germany) was used to identify any peptide cleavage of hGH following exposure to scCO2 and encapsulation. A small amount of each sample (Table 1) was dissolved in a 50:50 acetonitrile–water mixture (40 :L). The hGH solution (20 :L) was then added to a matrix solution (HCCA) (20 :L). This resultant mixture (0.5 :L) was spotted onto a MALDI-TOF plate and analysed in linear mode once the solvent had evaporated.
Human growth hormone was assayed and soluble aggregate formation was quantified by SEC as described in the British Pharmacopoeia27 using an Agilent 1100 series HPLC system (Agilent, Santa Clara, California). Briefly, each sample (Table 1) was dissolved in phosphate buffer (0.025 M, pH 7.0) to give a 1 mg/mL concentration, and this solution (20 :L) was injected into the system. A single mobile phase, comprising 97% phosphate buffer (0.063 M, pH 7.0) and 3% propan-2ol, was flowed through the column at 25◦ C at a flow rate of 0.6 mL/min. The column (30 cm × 7.8 mm TSKGel G2000SWXL ) (Tosoh Bioscience, Inc., San Francisco, California) was packed with a hydrophilic silica gel. The hGH peak eluted after approximately 15 min and was detected at a wavelength of 214 nm. Quantification of the percentage of monomer, dimer and oligomers present was determined by calculating the ratio of the area under the relevant peak as compared with the total area under the chromatogram. Size-exclusion chromatography was also used to determine the percentage of insoluble aggregates formed in each sample (Eq. 1):
(1)
Equation 1 gives the calculation of the percentage of insoluble aggregates by SEC: measured concentration, concentration given by SEC (mg/mL); expected concentration concentration in the original spraydried material (mg/mL). RP Chromatography Degradation of hGH via oxidation and deamidation was assessed using RP-HPLC, as described in the British Pharmacopoeia,27 with an Agilent 1100 series HPLC system (Agilent). Briefly, a butylsilyl silica gel column, 0.25 m long and 4.6 mm internal diameter (214TP54 column) (Grace-Vydac, Deerfield, Illinois), DOI 10.1002/jps
was fitted to an Agilent 1100 HPLC system (Agilent) and heated to 45◦ C. The column was washed with 50% acetonitrile–0.1% trifluoroacetic acid until the baseline was stable. The column was then equilibrated with a mobile phase of 71% 0.05 M Tris buffer–29% 1propanol at a flow rate of 1.5 mg/mL before injecting 20 :L of each sample (Table 1) containing 2 mg/mL hGH. Quantification of the percentage of native protein and mono-deamidated, di-deamidated and oxidised species present was determined by calculating the ratio of the area under the relevant peak as compared with the total area under the chromatogram.
IR Spectroscopy
Size-Exclusion Chromatography
Insoluble aggregates (%) Measured concentration × 100% =1− Expected concentration
59
Infrared spectroscopy was used to detect any alterations in the secondary structure of hGH as a result of exposure to scCO2 and the encapsulation process. Discs were made by grinding hGH (2 mg) with anhydrous KBr (200 mg) using a pestle and mortar. This mixture was pressed into a disc using a Specac press (Specac, Slough, UK). The discs were analysed using a Nicolet 210 IR spectrometer (Thermo Nicolet, Madison, Wisconsin) with 1048 scans and a resolution of 4 cm−1 . Each disc was analysed twice, and two were made for each condition in order to produce representative results. These four spectra were coadded, using an OMNIC software package (Fischer Scientific UK Ltd, Loughborough, UK), to produce an average. A blank disc was also produced and its spectrum subtracted from the samples. The secondary structures of the hGH samples were determined by analysing the second derivative of the spectra between 1600 and 1700 cm−1 .25
Far-Ultraviolet CD An Applied Photophysics Pi-Star-180 spectrophotometer (Applied Photophysics Ltd., Leatherhead, UK) was used to determine whether exposure to scCO2 or the encapsulation process caused any alterations to the secondary structure of hGH. Each sample (1.5 mg) was dissolved in sodium phosphate buffer (20 mM, pH 6.8) (300 :L) and diluted to give a final concentration of 10 :mol/dm3 . Spectra were obtained from 300 :L of sample in a 1-mm path length quartz cuvette. A blank spectrum was also recorded and subtracted from the hGH spectrum. The secondary structure was investigated by recording the spectra from 250 to 200 nm in 1 nm increments at 25◦ C. The temperature was regulated using a Neslab RTE-300 circulating programmable water bath and a thermoelectric controller (Melcor, Lawrence Township, New Jersey). The mean residue ellipticity was calculated (Eq. 2) and plotted in order to remove any concentration effects. Each condition (Table 1) was analysed in JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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duplicate. [2] =
2abs 10ncl
Table 2. Mass Spectrometry Analyses of hGH Following Exposure to scCO2 , High Temperatures and the Encapsulation Process
(2)
Equation 2 gives the calculation of the mean residue ellipticity from CD: [θ], mean residue ellipticity (deg cm2 /dmol); θobs , ellipticity (mdeg); n, number of amino acid residues in the protein; c, concentration of hGH (mol/dm3 ) as determined by HPLC SEC; and l, path length (cm). Statistical Analysis Statistical analyses were performed, using Microsoft Excel, following SEC and RP chromatography. Analyses were calculated for each condition (Table 1) using analysisof variance and least significant difference at 5% confidence level. Any differences in results were considered significant if the p value was less then 0.05.
RESULTS AND DISCUSSION The secondary globular structure of hGH is built up of a hydrophobic interior of four "-helices, surrounded by the remaining residues in the form of random chains and loops. The most common changes observed for hGH as a result of instability are secondary structural rearrangement, aggregation, oxidation and deamidation.20 As these may reduce the bioactivity of the protein and/or increase the possibility of side effects (particularly immunogenicity),21 it is essential that protein integrity is maintained upon encapsulation using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process of microparticle production. Hence, the stability of hGH was assessed using a range of analytical techniques following exposure to scCO2 and/or heat and after release from the microparticles. In the scCO2 exposure studies, the hGH was in contact with scCO2 for 1 h, as this is the length of time that the protein would be exposed during batch manufacture. hGH was successfully encapsulated into polymer microparticles using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process, generating a white free-flowing powder with an hGH encapsulation efficiency of 98.3 ± 4.6%. The protein was then extracted and stability was determined. These results were compared with an unexposed control of spray-dried hGH. The aim of these studies was to identify if the CriticalMixTM (Critical Pharmaceuticals Ltd.) process is suitable for the encapsulation of hGH and to determine the appropriate processing parameters (temperature and pressure). A number of samples for each condition were analysed and, with the exception of MALDI-TOF MS, the averages are presented. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
Conditions Temperature (◦ C)
CO2 Pressure (Bar)
Not exposed to scCO2 32 75 32 200 40 140 45 75 45 200 65 In air hGH encapsulated using the PGSS rig at 40◦ C and 140 bar
m/z hGH2+
hGH+
11,074 11,067 11,067 11,065 11,064 11,069 11,069 11,072
22,150 22,125 22,118 22,119 22,112 22,128 22,125 22,115
The absence of any additional peaks and no significant reduction in the m/z values shows that peptide cleavage has not occurred at the conditions analysed.
MALDI-TOF Mass Spectrometry Matrix-assisted laser desorption ionization–time-offlight MS analyses were performed in order to detect the cleavage of the peptide backbone upon exposure to scCO2 and the encapsulation process. hGH has a molecular weight of 22125 Da,20 and if no cleavage has occurred, it should display only peaks corresponding to this mass in the spectrum. The spectrum of hGH unexposed to heat and scCO2 (Fig. 1) displays two peaks, with m/z values of 11,074.1 and 22,150.1, corresponding to hGH2+ and hGH+ ions, respectively. The presence of only hGH+ and hGH2+ peaks at approximately 22,125 and 11,067, respectively, in each of the samples (Table 2) indicates that no fragmentation has occurred as a result of exposure to scCO2 or during the encapsulation process and that the scCO2 did not react in any way with the protein. If any peptide cleavage had occurred, additional peaks or a reduction in the m/z value over 56 (corresponding to the loss of one amino acid residue) would be observed. Cleavage of hGH has not been previously reported in the literature, as it is well known that cleavage is not a standard pathway of degradation.20 Clearly, from above, there are some slight discrepancies in the m/z values between the samples. These are caused by the presence of salts and impurities within the spray-dried material, and it is within the experimental error of the system.37 hGH Aggregation Aggregation is a common problem encountered with the processing of proteins when they are exposed to shear forces or liquid–air interfaces, leading to situations in which they can readily unfold.26 SEC was used to investigate the formation of dimers and higher-order aggregates following exposure to scCO2 at different temperatures and pressures and during the polymer encapsulation process. In SEC, the hGH DOI 10.1002/jps
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Figure 1. A typical MALDI-TOF MS spectrum of hGH unexposed to heat and scCO2 . The spectrum shows two peaks at 11074.1 and 22150.1 corresponding to hGH2+ and hGH+ ions, respectively.
monomer elutes at approximately 14.4 min and the dimer at 12.7 min. The control and the samples exposed to scCO2 , heat and the encapsulation process displayed a large peak at 14.4 min, indicating that the majority of each sample remained in the monomeric form (Fig. 2). Each of the samples exposed to scCO2 and the encapsulated hGH (red trace) were found to display a small dimeric peak which is comparable in size to that of the unexposed sample (blue trace). This indicates that no further aggregation had
taken place during the exposure. However, the samples exposed to 65◦ C (green trace) showed a marked increase in the size of the dimer peak. This is consistent with previous work2,34 in which high temperatures were observed to significantly increase the rate of aggregation as a result of enhanced mobility of the hGH. Integration of the monomeric peak of each sample (Table 3) shows no significant difference among the control, the encapsulated sample and the samples
Figure 2. Size-exclusion chromatography analysis to detect the formation of aggregates on exposure to scCO2 , heat and the encapsulation process. The arrows indicate the expected retention time of the hGH dimer. Equivalent peaks were observed for the unexposed sample (blue trace) and hGH exposed to 40◦ C and 140 bar (red trace), showing that no aggregation had occurred. A larger peak was observed at 65◦ C (green trace), showing the formation of the dimer. DOI 10.1002/jps
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Table 3.
Calculation of the Percentage of Monomer Remaining Within Each Sample Conditions
Temperature
(◦ C)
CO2 Pressure (Bar)
Not exposed to scCO2 32 75 32 200 40 140 45 75 45 200 60 75 65 In air hGH encapsulated using the PGSS rig at 40◦ C and 140 bar Significant difference 5% LSD
SEC Results Monomer (%)
Dimer (%)
Oligomers (%)
99.1a
0.9a
99.0a 98.6 98.7a 98.4 97.7 97.9a 96.2 99.3a
1.0a 1.4 1.2a 1.5 1.9 1.3a 3.4 0.7a
0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.3 0.0
p = 0.009 0.71
p = 0.052 0.47
– –
Some monomeric hGH is lost on exposure to 65◦ C as a result of aggregate formation. a As a result of a limited supply of hGH, statistics could only be performed on these samples. LSD, least significant difference.
exposed to scCO2 below 45◦ C, proving that hGH is stable to aggregation at these conditions. This highlights the advantage of scCO2 processing, when encapsulating proteins into polymers for sustainedrelease drug delivery applications, over alternative encapsulating processes such as emulsification and spray drying, which can generate up to 41% aggregation of hGH.1,3,38 This is expected, as aggregation generally occurs in the presence of liquid–air and water–oil interfaces as a result of unfolding of the protein. As the CriticalMixTM (Critical Pharmaceuticals Ltd.) process encapsulates proteins as a dry powder and is carried out in a CO2 atmosphere, these interfaces do not exist and, therefore, the protein does not unfold and aggregate. Significant reductions in the monomeric content (p = 0.009), coupled with an increase in dimer and oligomers formation, were observed upon increasing the temperature further (Table 3). As discussed previously, this is as a result of the increased mobility of the protein at higher temperatures.2,34 No significant reduction in the monomeric content was observed on increasing the scCO2 pressure. Furthermore, it was shown that hGH did not undergo aggregation when encapsulated into the microparticles at 40◦ C and 140 bar. Comparison of the expected (i.e., protein content in the spray-dried powder measured prior to encapsulation or exposure to scCO2 ) and hGH concentration after encapsulation or exposure to scCO2 permits assessment of the formation of insoluble aggregates that would be unavoidably excluded from the SEC analysis. Insoluble aggregates are thought to form, initially, as soluble aggregates which then act as nucleation sites for further aggregation until the resulting aggregate becomes insoluble.39 Figure 3 shows the results for hGH exposed to 75 bar scCO2 at various temperatures. No significant difference (p = 0.132) was observed in insoluble aggregation formation between JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
the four temperatures and the control. This indicates that in the solid state, the spray-dried hGH was surprisingly stable to the temperatures tested over the 1-h exposure period. Although not significantly different, an increase in the percentage of the insoluble aggregates was observed at 90◦ C. This is consistent with the increase in dimer formation observed on raising the temperature, highlighting that aggregation is temperature dependent. The formation of insoluble aggregates is important, as it would reduce the bioavailability of the protein and could, therefore, have a negative impact upon efficacy. Secondly, the insoluble aggregate could be more immunogenic and may increase the risk of patients generating neutralising antibodies to hGH or other side effects.
hGH Oxidation and Deamidation Oxidation of proteins can cause a partial or complete loss in the bioactivity,28 whereas deamidation can affect the conformation of the protein,29 and it occurs either in an aqueous environment or in the solid state as a result of the presence of moisture. RP chromatography was used to investigate the oxidation and deamidation of hGH following exposure to scCO2 and/or heat and encapsulation into polymeric microparticles. Integration of the main hGH peak gives the percentage of remaining native hGH. Close examination of the results obtained for various conditions (Table 4) reveals no significant change on altering the scCO2 pressure, but a significant reduction (p = 0.047) in the native hGH content upon increasing the temperature. It is well established that the extent of deamidation of hGH is temperature dependent.2,34 Furthermore, hGH encapsulated using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process at 40◦ C and 140 bar did not undergo oxidation or deamidation (Table 4). DOI 10.1002/jps
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Figure 3. Assessment of insoluble aggregate formation of hGH exposed to CO2 at 75 bar and various temperatures. No significant difference was detected among the four temperatures (p = 0.132).
Further examination of the chromatograms used to calculate these results provides information on the source of this degradation (Fig. 4 and Table 4). In a degraded sample, the hGH native peak is eluted after approximately 33.5 min, and the mono-deamidated, di-deamidated and oxidised peaks appear prior to it at 27.5, 23 and 25 min, respectively.30 The control (blue trace) and hGH exposed to 40◦ C at 140 bar (red trace) show the presence of a small peak at approximately 27 min, which becomes more pronounced at 65◦ C (green trace), indicating that hGH degrades principally by deamidation. Integration of these peaks
(Table 4), although not significant, shows a general trend of increasing mono-deamidation on raising the temperature, with the change in pressure appearing to have little effect. This mirrors the earlier work which reported that hGH can deamidate when stored at high temperatures.34 A peak at 23 min also becomes distinctive at 65◦ C, indicating that hGH undergoes both mono- and di-deamidation at high temperatures. Previous work has showed asparagine-149 to be the principal site for deamidation, with a slower reaction also occurring at asparagine-152.40,41 This was attributed to these residues being situated within
Table 4. Calculation of the Percentage of Native Protein, Mono-Deamidated, Di-Deamidated and Oxidised Species Present after Processing Conditions Temperature (◦ C)
CO2 Pressure (Bar)
Not exposed to scCO2 32 75 32 200 40 140 45 75 45 200 60 75 65 In air hGH encapsulated using the PGSS rig at 40◦ C and 140 bar Significant difference 5% LSD
RP Results Native hGH (%)
Mono-Deamidated (%)
Di-Deamidated (%)
Oxidated (%)
97.4a
1.9a
0.1a
97.8a 96.3 96.9a 95.9 95.1 96.2a 92.8 98.2a
1.6a 2.4 2.4a 2.4 3.5 2.7a 4.8 1.1a
0.1a 0.8 0.1a 0.7 0.6 0.4a 1.0 0.0a
0.5a 0.4a 0.4 0.5a 0.9 0.3 0.5a 1.3 0.4a
p = 0.047 1.33
p = 0.073 1.19
p = 0.33 0.33
p = 0.84 0.34
A significant loss is observed upon increasing the temperature, although this is not reciprocated in the encapsulated hGH. A much greater loss (4%) occurs upon exposure to 65◦ C. a As a result of a limited supply of hGH, statistics could only be performed on these samples. LSD, least significant difference.
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Figure 4. RP analysis to detect the presence of degradation products upon exposure to scCO2 and/or heat. The ↓, and ∗ represent the expected retention times of mono-deamidated, dideamidated and oxidised products, respectively. An increase in the intensity of the deamidation peaks was observed at 65◦ C, showing that high temperatures are capable of degrading hGH.
a flexible, exposed loop of the protein, making them easily accessible. It is likely, therefore, that at low temperatures, only asparagine-149 deamidates, but upon increasing the temperature, asparagine-152 also becomes susceptible.33,41
Oxidation was not observed when in the presence of CO2 . This is expected, as either hydrogen peroxide, molecular oxygen or organic solvents must be present for the amino acid residues to be oxidised.29 This analysis highlights another key advantage of scCO2
Figure 5. CD analysis following exposure to scCO2 . The similarity in the shape of each of the exposed samples to the control sample demonstrates that no change has occurred in the secondary structure. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 1, JANUARY 2012
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processing over conventional encapsulation methods, as organic solvents are not required and, therefore, degradation is reduced. Far-Ultraviolet CD Assessment of the conformation and stability of hGH was performed by measuring the CD properties of spray-dried hGH and hGH exposed to heat, scCO2 and the encapsulation process. CD has been previously used to probe the secondary structure of hGH in solution, resulting in two strong minima at 209 and 221 nm, indicative of an "-helical structure.20 The CD results obtained in the present study (Fig. 5) are consistent with this theory, confirming the secondary structure of hGH to be "-helical. Each of the samples analysed, including hGH exposed to high temperatures, showed similar "-helical traces, demonstrating that scCO2 and/or heat and the encapsulation process do not alter the secondary structure of hGH. This was expected, as previous work has shown the secondary structure of hGH to be stable in the solid state to temperatures in excess of 150◦ C.42 Each sample displays different absorbances; however, as the ratio between the two troughs (with the exception of the extracted sample) remains consistent, this is not indicative of a change in the secondary structure. As small concentrations are required for CD as compared with SEC, these differences in absorption are likely to be generated by the experimental error during the dilution from bulk SEC samples. A slight deviation was observed in the magnitude of
65
the peak at 221 nm for the extracted sample. This increase would suggest a greater "-helix structure; however, the change is only slight and, therefore, this is not conclusive. Many researchers have shown that a partial loss of hGH "-helix content, coinciding with an increase in the $-sheets, can occur as a result of solvent exposure during encapsulation by emulsion and spraydrying techniques as a result of exposure to liquid–air interfaces.3,25 Therefore, these data again highlight a significant advantage of using scCO2 processing to encapsulate proteins for controlled-release drug delivery, as solvents are not required and, therefore, no liquid–air or oil–water interfaces are present. IR Spectrometry The second derivative of an IR spectrum provides information about the secondary structure of proteins in the solid state,1,43 and has the additional advantage that samples can be measured in the solid state. These data can be used to detect any structural rearrangements caused by exposure to scCO2 and the encapsulation process. The second derivative of the IR spectrum of hGH (Fig. 6) shows four discrete peaks at approximately 1640, 1654, 1683 and 1695 cm−1 . These are indicative of extended loop structures, an "-helix and a $-turn, respectively.44 The intensities of these peaks provide qualitative information about the ratios of these structures within hGH. The similarity of these four peaks among the unexposed, scCO2 and/or heat exposed and extracted sample shows that
Figure 6. The second derivative of the IR spectrum between 1600 and 1700 cm−1 , illustrating that no alteration occurs in the secondary structure of hGH as a result of exposure to scCO2 and the encapsulation process. DOI 10.1002/jps
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no alterations have occurred in the secondary structure. Each of the conditions analysed displayed similar traces. These results are consistent with those obtained by CD, proving that the secondary structure of hGH is stable to scCO2 , heat and the encapsulation process.
CONCLUSION This work has shown that hGH can be successfully encapsulated within polymers, using an encapsulation method based on exposure of polymers to scCO2 , without degrading or denaturing the hGH. MALDI-TOF MS analyses demonstrated that no peptide cleavage occurred as a result of exposure to scCO2 or during the encapsulation procedure, and that the scCO2 did not react with the protein. CD and IR analyses concluded that the secondary structure remained unaltered during CO2 exposure and encapsulation. SEC and RP demonstrated that both aggregation and deamidation were influenced by temperature but not pressure. SEC showed the occurrence of aggregation at 65◦ C resulting in a loss in the hGH monomer content of 2.5%. No significant aggregation was observed following exposure to scCO2 below 45◦ C or the encapsulation process. RP chromatography revealed that the extent of deamidation increased upon raising the temperature. Exposure to 65◦ C showed the occurrence of both mono- and di-deamidation, illustrating that both asparagine 149 and 152 are deamidated. Deamidation was not observed following the encapsulation procedure, ascertaining that the polymers and excipients present in the spray-dried material are able to protect the protein. Comparison of these results to similar studies following other encapsulation methods has highlighted the enhanced stability when processing using the CriticalMixTM (Critical Pharmaceuticals Ltd.) process to encapsulate proteins in polymers for injectable sustained-release drug delivery applications.
ACKNOWLEDGMENTS This work was funded by the Engineering and Physical Sciences Research Council and Critical Pharmaceuticals Ltd. The authors also gratefully acknowledge Professor Mark Searle for his expertise and advice with CD. Thanks also to Mr Peter Fields, Mr Richard Wilson and Mr Paul Gaetto of University of Nottingham technical workshop.
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