Pharm Res (2012) 29:3278-3291
`DOI 10.1007/sl 1095-012-0820-7
`
`Effects of Excipients on the Chemical and Physical Stability of Glucagon
`during Freeze-Drying and Storage in Dried Formulations
`
`Wei-Jie Fang• Wei Qi· John Kinzell ·Steven Prestrelski ·John F. Carpenter
`
`Received: 15 December 2011 I Accepted: 22June 2012 /Published online: 6.July 2012
`CCl Springer Science+ Business Media, LLC 2012
`
`ABSTRACT
`Purpose To evaluate the effects of several buffers and exci(cid:173)
`pients on the stability of glucagon during freeze-drying and
`storage as dried powder formulations.
`Methods The chemical and physical stability of glucagon in
`freeze-dried solid formulations was evaluated by a variety of
`techniques including mass spectrometry (MS), reversed phase
`HPLC (RP-HPLC). size exclusion HPLC (SE-HPLC). infrared (IR)
`spectroscopy, differential scanning calorimetry (DSC) and turbidity.
`Results Similar to protein drugs, maintaining the solid amor(cid:173)
`phous phase by incorporating carbohydrates as well as addition
`of surfactant protected lyophilized glucagon from degradation
`during long-term storage. However, different from proteins,
`maintaining/stabilizing the secondary structure of glucagon was
`not a prerequisite for its stability.
`Conclusions The formulation lessons learned from studies of
`freeze-dried formulations of proteins can be applied successfully
`to development of stable formulations of glucagon. However,
`peptides may behave differently than proteins due to their small
`molecule size and less ordered structure.
`
`KEY WORDS excipients · freeze-drying · peptides · solid
`states · stability
`
`W.-J. Fang· W. Qi · j. F. Carpenter(~)
`Department of Pharmaceutical Sciences
`University of Colorado Denver
`Aurora, CO 80045, USA
`e-mail: john.Carpenter@ucdenver.edu
`
`J. Kinzell · S. Prestrelski
`Xeris Pharmaceuticals, Inc.
`San Rafael, CA 9480 I, USA
`
`Present Address:
`W.-J. Fang
`Zhejiang Hisun Pharmaceutical Inc.
`46 Waisha Rd, Jiaojiang District,
`Taizhou, Zhejiang 3 I 8000, China
`
`~Springer
`
`ABBREVIATIONS
`Abbreviations used for amino acids follow the rules of the IUPAC(cid:173)
`IUB Joint Commission of Biochemical Nomenclature (Eur. j. Bio(cid:173)
`chem. 1984 138, 9-37)
`CD
`cyclodextrin
`differential scanning calorimetry
`DSC
`ESl-MS
`electrospray ionization-mass spectrometry
`HES
`hydroxylethyl starch
`IR
`Infrared
`PES
`polyethylstyrene
`PS
`polysorbate
`RP-HPLC
`reversed-phase high-performance liquid
`chromatography
`size exclusion
`glass transition temperature
`retention time
`
`SE
`Tg
`tR
`
`INTRODUCTION
`
`Peptides have become increasingly important as therapeutic
`products. Currently there are more than 60 approved pep(cid:173)
`tide drugs, and an additional 130 peptide candidates are in
`clinical development (1). As is the case with protein thera(cid:173)
`peutics, peptide drugs are susceptible to both chemical and
`physical degradation, and stabilization or these products is
`challenging (2-5). One approach to achieving suflicient
`stability of a biological product is to develop a freeze-dried
`formulation (6, 7). There is an extensive literature describing
`the capacities of various excipients to stabilize proteins dur(cid:173)
`ing the freeze-drying process and long-term storage in dried
`formulations, as well as the mechanisms for such stabiliza(cid:173)
`tion (6--8). For peptide drugs there is a more limited litera(cid:173)
`ture on stabilization in freeze-dried formulations (9-12).
`Therefore, the goal of the current study was to gain forther
`NPS EX. 2049
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`IPR2015-0 I 093
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`

`
`Effects of Excipients on Glucagon Stability in Solid
`
`insights into how the lessons learned from protein therapeu(cid:173)
`tics apply to development of stable freeze-dried formulations
`of peptide drugs.
`Typically, an essential stabilizer in a freeze-dried protein
`formulation is a nonreducing disaccharide such as sucrose or
`trehalose (6, 7). These sugars can inhibit protein unfolding
`dming the freezing and drying steps of freeze-drying, as well
`as provide a glassy matrix that is important for long-term
`storage stability of the dried product (6, 7). For drugs that are
`formulated at acid pH, sucrose has the disadvantage of
`being susceptible to acid-catalyzed hydrolysis forming re(cid:173)
`ducing sugars glucose and fructose,( 13) which can chemical(cid:173)
`ly degrade proteins or peptides via the l\!Ialliard reaction
`( 13). For example, the Malliard reaction has been reported
`when glucagon was formulated with the reducing disaccha(cid:173)
`ride lactose ( 14'). Trehalose is more resistant to acidic hy(cid:173)
`drolysis than sucrose and is more suitable for formulations at
`low pH (15,16).
`The inclusion of a polymer such as hydroxylethyl starch
`(HES) to a lyophilized protein formulation may improve
`long-term storage stability of the protein in formulations
`that also contain sucrose or trehalose (17). The increased
`stability is observed because HES can form glassy matrix
`with very high T g (i.e. >200°C). However, HES alone
`usually fails to confer stability to dried proteins because it
`does not inhibit protein unfolding during freeze-drying, and
`the rate of degradation during storage is greatly increased
`for unfolded proteins (6, 7, 1 7).
`The non-ionic surfactants polysorbate 20 and 80 have been
`used extensively as excipients in freeze-dried and aqueous
`solution formulations of proteins due to their ability to reduce
`protein aggregation (18-20). This protective effect ha5 been
`att1ibuted to several different mechanism5 including compet(cid:173)
`ing with protein molecules for interfaces, increasing thermo(cid:173)
`dynamic stability of the native state through binding to the
`protein, fostering refolding and reducing the concentration of
`protein molecules in a stagnant boundary during rehydration
`of dried formulations (20). Cyclodextrins also have been
`shown to reduce protein and peptide aggregation by compet(cid:173)
`ing for the air-water interface (21) and by binding to the
`hydrophobic residues of proteins or peptides such as glucagon
`(14,,22).
`The potential stabilizing effects of each of these classes of
`protein stabilizers for small peptides in freeze-dried formu(cid:173)
`lations are not well understood. Among therapeutic pepti(cid:173)
`des, insulin has been most studied in dried formulations.
`Altl1ough insulin is different from most other peptides in
`that it has relatively well-defined tertiary and quaternary
`structures, it is still instructive to consider results for it in the
`broader context of peptide stabilization. Previous work has
`shown that insulin in freeze-dried formulations exhibited
`many of the same pathways of degradation (i.e. aggregation
`and deamidation) as in aqueous solution (9, 10). In one
`
`,<
`
`3279
`
`study, t11e degradation rate was dependent on the pH of
`aqueous solution p1ior to freeze-drying as well as the water
`content of the freeze-dried cake (9,10). Covalent dimeriza(cid:173)
`tion of human insulin was substantially decreased by incor(cid:173)
`poration into a glassy matrix of trehalose, presumably by
`inhibiting structural perturbation of the peptide, reducing
`molecular mobility in the dried formulation and physically
`separating the insulin molecules ( 10). In another study,
`insulin freeze-dried with trehalose exhibited a substantially
`lower local dynamics (P-relaxation) and lower degradation
`rates (i.e. deamidation and dimerization) than insulin freeze(cid:173)
`dried with dextran ( 12). The authors speculated P-relaxation
`of insulin was reduced because of hydrogen bonding 'vith
`trehalose, and thus chemical degradation was inhibited. On
`the other hand, dextran cannot form hydrogen bonds with
`insulin as readily due to steric hindrance, and therefore
`reduction of P-relaxation was not present in the dextran
`formulation.
`The study of stability in freeze-dried formulation with
`other peptides is rather limited. For example, in one study,
`the level of mannitol freeze-dried formulations of atrial
`natriuretic peptide (ANP) affected the amount of multimers
`formed during storage. The authors speculated that in the
`less stable formulations, which had higher mannitol levels,
`mannitol crystalized and increased ilie water moisture con(cid:173)
`tent in the amorphous phase ( 11 ).
`Glucagon (Fig. 1 ), the focus of the current study, is a
`polypeptide hormone composed of 29 amino acid residues
`that is currently used for the emergency treatment ofinsulin(cid:173)
`induced hypoglycemia (23). Glucagon is known for its pro(cid:173)
`pensity to degrade both chemically (i.e., hydrolysis and
`oxidation)(24-26) and physically (i.e., aggregation)(27 ,28);
`including formation of aggregates during freeze-drying and
`rehydration in the absence of stabilizing excipients (29).
`Therefore, it is an excellent model peptide to evaluate the
`effects of different buffers, the surfactant polysorbate 20,
`trehalose, HES and P-CD on stability during freeze-drying
`and storage in dried formulations.
`
`MATERIALS AND METHODS
`
`Materials
`
`All chemicals were of reagent grade or higher quality. Glu(cid:173)
`cagon was purchased from the American Peptide Company
`(Sunnyvale, CA). P-CD was purchased from Cyclodext1in
`Technologies Development, Inc. (High Springs, FL). Tre(cid:173)
`halose dihydrate was purchased from J. T.Baker (Philips(cid:173)
`burg, NJ). HES (Viastarch, MW 200KDa) was purchased
`from Fresenius (Graz, Austria). Phosphoric acid, citric acid,
`glycine, 2M hydrogen chloride, sodium chloride, sodium
`hydroxide, acetonitrile, hydrogen peroxide, potassium
`
`~Springer
`
`Page2
`
`

`
`3280
`
`Fang et al.
`
`His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp9-Tyr-Ser-Lys-Tyr-Leu-Asp 15 -Ser-Arg-Arg-Ala-Gln-
`
`Fig. I Primary structure of glucagon. The three Asp and Met residues are shown in bold.
`
`Asp21 -Phe-Val-Gln-Trp-Leu-Met27-Asn-Thr
`
`bromide and polysorbate 20 were purchased from Fisher
`Scientific (Hampton, NH). Nylon membrane filters
`(0.4·5 µm) were purchased from Whatman (Maidstone, Eng(cid:173)
`land). Polyethylstyrene (PES) membrane filters (0.2 µm)
`were purchased from Millipore (Billerica, MA). 3-ml lyoph(cid:173)
`ilization vials (borosilicate glasses, type 8412-B) and gray
`butyl stoppers (Product# 10123524) were purchased from
`West Pharmaceutical (Lionville, PA). Other reagents and
`chemicals were purchased from Sigma-Aldrich (Jvlilwaukee,
`WI).
`
`Acidic Degradation of Glucagon in Aqueous Solution
`
`Glucagon (0.5 mg/ml) was dissolved in 5 mM sodium phos(cid:173)
`phate bufler (pH=3.0) and incubated for 5 days at 60°C.
`Aliquots (0.5 mL) of the solution were removed at various
`times and centrifoged at 14,500 rpm for 10 min. The
`supernatant was analyzed by reversed-phase high(cid:173)
`performance liquid chromatography (RP-HPLC) as de(cid:173)
`scribed below. Samples were also analyzed by electrospray
`ionization-mass spectrometry (ESI-MS; see below).
`
`Hydrogen Peroxide-Induced Oxidation of Glucagon
`in Aqueous Solution
`
`We followed the USP method (USP 30 Official Monograph
`for Glucagon, 2007) for forced oxidation of glucagon. Glu(cid:173)
`cagon (1 mg/ml) was oxidized by incubation in 0.6% hy(cid:173)
`drogen peroxide (H20 2), 20% acetonitrile and 80% water
`with 10 mM HCl at 4°C for 210 min. Aliquots were re(cid:173)
`moved at various times and analyzed by RP-HPLC using
`the method described below. The oxidation products were
`also analyzed by ESI-MS as well as tandem MS (MS-MS;
`see below).
`
`ESl-MS and MS-MS Analysis of Glucagon
`and its Degradation Products
`
`temperature. For MS-MS experiment~ on glucagon and
`oxidized glucagon, various ml z components were selected
`and fragmented with suitable collision energy to have rea(cid:173)
`sonable amount of peaks following fragmentation.
`
`Sample Preparation for Freeze-Drying
`
`Glucagon (10 mg/mL) was dissolved in three different buf(cid:173)
`fers (glycine hydrochloride, sodium phosphate, and sodium
`citrate buflers, 5 mM, pH 3.0). The solution was then mixed
`in a l: l (v /v) ratio with various excipient solutions (prepared
`at twice the desired concentration using corresponding bufl~
`er) to obtain a final glucagon concentration of 5 mg/mL and
`the final desired excipient concentration of 0.0 l % for poly(cid:173)
`sorbate 20 and 10 mg/mL for the carbohydrates. The
`solution was then filtered through 0.2 µm Millipore PES
`membrane. Sample preparation was conducted in a 4°C
`cold room. The glucagon concentration and the purity were
`determined by RP-HPLC (see below).
`
`Freeze-Drying
`
`The formulations were pipetted (0.3 mL) into 3-ml lyophiliza(cid:173)
`tion vials (13-mm ID) and freeze-dried in a FTS Durastop
`freeze-drier (Stoneridge, NY). For freezing, samples were
`cooled to -40°C at 2.5°C/min and maintained at this tem(cid:173)
`perature for 2 h. Then the shelf temperature was increa~ed to -
`5°C at 2°C/min and held for 2 has an annealing step (29). The
`temperature was then decreased to -30°C at l.5°C/min and
`the chamber pressure was reduced to 8 Pascal. These condi(cid:173)
`tions were maintained during 24 h for primary drying. Then
`the shelf temperature was increased to 40°C at 0.5°C/min and
`held at 40°C for l 0 h for secondary drying. Then, the vials
`were stoppered under vacuum using gray butyl stoppers. None
`of the formulations showed any visual evidence of cake collapse
`following freeze-drying.
`
`Storage Studies
`
`Glucagon and its degradation product~ were analyzed with
`an electrospray-triple quadrupole-time-of~flight mass spec(cid:173)
`trometer (ESI-qTOF-MS) from Applied Biosystems (PE
`SCIEX/ ABI API QSTAR Pulsar i Hybrid LC/MS/
`MSESL). Mass Spectra were acquired by scanning a mass(cid:173)
`to-charge ratios (ml z) range from l 00 to 2000. Eluates
`(l mg/ml glucagon, 50 µl) were injected into the mass
`spectrometer at a flow rate of 5 µI/min. Spray voltage wa~
`set at 4500 V, and capillary temperature was set at ambient
`
`Following freeze-drying, the sample vials were incubated at
`60°C and analyzed after 2 weeks. Triplicate sample vials for
`each formulation were incubated. Control sample vials
`(equivalent to time zero of incubation) were stored at -80°
`C until analysis. Following storage, formulations were first
`rehydrated to 5 mg/mL with water and then diluted to
`l mg/mL glucagon with the corresponding buffer. An ali(cid:173)
`quot of the rehydrated sample (200 µL) was analyzed for
`
`~Springer
`
`Page 3
`
`

`
`Effects of Excipients on Glucagon Stability in Solid
`
`3281
`
`turbidity (see below). Then, the solution was centrifoged
`(14·,500 111mx 10 min) to removed insoluble material. The
`supernatant was diluted 1: 1 with the corresponding buffer
`for RP-HPLC and size exclusion (SE)-HPLC analysis (see
`below).
`
`Differential Scanning Calorimetry (DSC)(JO)
`
`The gJa5s transition temperature (Tg) of dried formulations
`was determined using a Perkin-Elmer Pyris-1 DSC (Nor(cid:173)
`walk, CT). Prior to sample analysis, the instrument was
`calibrated for melting temperature and heat of fusion with
`indium (onset of melting: 156.6°C; heat of fusion: 28.4-5 JI
`g). In a d1y box, the sample vials were opened and about
`1 mg samples of dry powder were placed in hermetically
`sealed aluminum pans. DSC thermograms were collected
`from 25°C up to 250°C (depending on the compositions of
`formulations) at a heating rate of 100°C/min. Baselines
`were determined using an empty pan, and all thermograms
`were baseline corrected. For measurement of T g> the sam(cid:173)
`ples were first heated to above T g to remove thermal histo1y,
`and then cooled back to 25°C and rescanned at 100°C/min.
`The thermogram obtained during the second scan was used
`to measure T g which was determined as the midpoint of the
`transition.
`
`Infrared (IR) Spectroscopy
`
`Infrared spectra of glucagon in aqueous solution and in
`freeze-dried formulations were collected at room tempera(cid:173)
`ture using a Bomem Prata spectrometer (Quebec, Canada)
`purged with d1y nitrogen (31,32). Spectra of glucagon in
`aqueous solution were obtained at a peptide concentration
`of 20 mg/ml (5 mM sodium phosphate buffer, pH 3.0) in a
`6 µm pathlength cell with CaF2 windows. For each sample,
`a 32-scan interferogram was collected in the single-beam
`mode with a resolution of 4 cm - t. Peptide spectra were
`processed to substract absorbance from water vapor and
`the buffer spectrum as previously described (31,32). For IR
`spectra of freeze-dried samples, a mass of dried formulation
`containing about 1 mg of glucagon was mixed with 500 mg
`potassium bromide powder. The mixture was ground gently
`and then annealed into a pellet using a hydraulic press (30).
`The spectra were transformed to second derivatives using
`Bomem Grams/32 software. The final protein spectra were
`smoothed with a seven-point Savitsky-Golay function. For
`comparison of spectra they were area normalized in the
`amide I region ( 1600-1700 cm -l) and overlaid (30).
`
`using a Molecular Devices microplate reader (Sunnyvale,
`CA). The freeze-dried glucagon formulations were first
`rehydrated with water to 5 mg/mL glucagon and then
`diluted with corresponding buffer to 1 mg/mL peptide
`concentration. Prior to analysis, the plates were shaken for
`10 s to mix the solutions in the wells.
`
`Reversed Phase-High Performance Liquid
`Chromatography (RP-HPLC)
`
`Chemical degradation of glucagon formulations was quan(cid:173)
`tified by RP-HPLC, using an Agilent 1100 HPLC system
`(Agilent Technologies, Inc., vValdbronn, Karlsruhe, Ger(cid:173)
`many) with a Thermo Biobasic CS column (5 µm, 250 x
`4.0 mm ID, Waltham, MA) and a Restek VWD Gl3J4.A
`detector (Bellefonte, PA). The mobile phase was 73%
`phosphate-cysteine bufler (pH 2.6) with 27% acetonitrile
`(USP 30 Official Monograph for Glucagon, pp 2230-
`2231, 2007). Before use, mobile phase was filtered with
`0.4·5 µm Nylon membrane filters and degassed. The flow
`rate for the analysis was 1 ml/min, and elution was moni(cid:173)
`tored at 214 nm. The temperature of the column was
`maintained at 37°C. The peak areas of glucagon and its
`chemical degradation products were used to determine the
`chemical degradation of glucagon occurring during freeze(cid:173)
`drying and storage.
`
`Size Exclusion-HPLC (SE-HPLC)
`
`Aggregation of glucagon in different formulations was de(cid:173)
`termined with SE-HPLC, using an Agilent 1100 HPLC
`equipped with a TSK G2000SWx1 gel filtration column
`(5 µm, 300X 7.8 mm ID) and a Restek DAD Gl315A
`detector. The mobile phase was 3.2 mM HCl, 100 mM
`sodium chloride, pH 2.5. Before use, the mobile phase was
`filtered and degassed. The flow rate for the analysis was
`1 ml/min and elution was monitored at 280 nm. The
`percent of monomer remaining in formulations was calcu(cid:173)
`lated based on the monomer area of the formulation sam(cid:173)
`ples compared to that for liquid control sample. Soluble
`aggregates were at minimal levels (<0.5%) in all of the
`samples (data not shown).
`
`RESULTS AND DISCUSSION
`
`Accelerated Acid Degradation of Glucagon
`in Aqueous Solution
`
`Optical Density at 405 nm (OD40s)
`
`The presence of aggregates in rehydrated formulations
`(1 mg/mL glucagon) was assessed by measuring the OD405
`
`Glucagon has an isoelectric point of about 7 and has a high
`solubility at pH values less than 3 or greater than 9. The
`recommended pH range for solutions of the peptide is
`between 2.5 and 3.0 (US pharmacopeia 24, 1999).
`
`~Springer
`
`Page4
`
`

`
`3282
`
`Fang et al.
`
`However, as in the case with many peptide and protein
`drugs, glucagon is susceptible to acid-catalyzed degradations
`(i.e. Asp cleavage) (24-26). To develop an assay to charac(cid:173)
`terize acid-catalyzed hydrolysis of glucagon, the peptide was
`first incubated in 5 mM sodium phosphate buffer (pH=3.0)
`at 60°C and characterized as a fonction of incubation time.
`The commercial glucagon we used had some impurities
`prior to incubation (about 8-9%, see Fig. 3a and its en(cid:173)
`larged version Fig. 3b). During incubation, glucagon under(cid:173)
`went significant further chemical degradation, with 50%
`loss of the parent peptide after incubation for 5 days
`(Figs. 2 and 3d). As expected in the acidic conditions,
`the main degradation products were due to Asp cleavage,
`as confirmed by ESI-MS analysis (Table I). Glucagon has
`three Asp residues and, therefore, six main fragments were
`observed in ESI-MS.
`In RP-HPLC (Fig. 3d), four main peaks (retention time
`(tR) around 7, 14, 19 and 29 min, respectively) and several
`smaller peaks (i.e. lR around 33, 37, 40, and 42 min) were
`observed. The main peaks were probably due to Asp cleav(cid:173)
`age whereas the smaller peaks could be due to Asp cleavage
`and/or deamidation (25,26). Glucagon has three Gln and
`one Asn residue. Deamidated residues were not specifically
`identified in the current study.
`
`Accelerated Oxidation of Glucagon in Aqueous
`Solution
`
`Although oxidation of Met does not impact the binding
`aflinity of glucagon to its receptor (33), the potency the
`oxidized peptide in stimulating glucose production in isolat(cid:173)
`ed rat adipocytes and hepatocytes is decreased (34-). There(cid:173)
`fore, oxidation of the peptide should be monitored and
`minimized. To develop an assay for oxidized glucagon, the
`peptide was incubated in 0.6% (v/v) hydrogen peroxide at
`room temperature (Fig. 4). Samples were removed as a
`function of time and analyzed by RP-HPLC. Two new
`
`'[ 2.0
`
`Cl ns u a 1.9
`Cl c ·c: 1.8
`"itj
`~
`0:: 1.7 c
`.3
`
`Cl
`
`0
`
`4
`2
`Time (days)
`
`6
`
`Fig. 2 Degradation of glucagon in 5 mM sodium phosphate buffer pH 3.0 at
`60°C. The % remaining glucagon was determined from chromatograms
`obtained by RP-HPLC. Error bars indicating SD are smaller than the symbols.
`
`~Springer
`
`peaks with similar tR and almost identical areas were ob(cid:173)
`served (tR around 8-9 min, Fig. 3c). Almost all of the parent
`peptide had decomposed after 210 min of incubation
`(Fig. 4). The oxidation products had molecular weights of
`[Glucagon+ 16] (calculated mass=3498.6; observed: [M +
`2 H] 2+=1750.2, [M+3 H] 3+=1167.l, [Iv1+4 H] 4+=875.6,
`[M +5 HJ 5+=700.7), suggesting one residue of glucagon was
`oxidized. MS/MS analysis (Table II) of the oxidation prod(cid:173)
`ucts showed the Met residue at position 27 was oxidized.
`The two peaks in the RP-HPLC chromatograms were prob(cid:173)
`ably due to two diastereomers formed with oxidation of the
`Met residue. Even though glucagon also has several other
`amino acids susceptible to oxidation (e.g. His, Trp, Tyr, and
`Phe), no degradation of these residues by hydrogen peroxide
`was observed.
`
`Effects of Buffers and Excipients on Glucagon's
`Stability in Freeze-Dried Formulations
`
`To determine formulation effects on stability of freeze-dried
`glucagon formulations, we evaluated effects of three hullers
`(glycine hydrochloride, sodium phosphate and sodium cit(cid:173)
`rate bullers, 5 mM, pH 3.0), polysorbate 20 and three
`carbohydrate excipients, trehalose, HES and P-CD. An
`accelerated degradation condition (storage at 60°C for
`2 weeks, followed by rehydration) was used to evaluate the
`stability of the peptide in the freeze-dried formulations.
`Important physical properties (peptide secondary structure
`and T g) were also characterized for each freeze-dried
`formulation.
`The T g of the freeze-dried formulations was determined
`by DSC (Table III). When there was no excipient, the Tg of
`dry powders was between l 22°C and l 4·0°C, depending on
`the buffer used in formulation (Table III). A~ expected, the
`presence ofpolysorbate 20 (0.01%w/v) did not have much
`effect on Tw
`For formulations with glass forming carbohydrates, the
`Tg was highest for HES and P-CD, followed by the mixture
`of trehalose and HES, and then trehalose alone (Table III).
`These values were consistent with the T g of the
`corresponding pure carbohydrates and their mass ratios in
`the formulations. The huller also had a substantial ellect on
`T g' especially in the cases where HES and P-CD were used
`as excipients (Table III). The cause(s) of the huller effect was
`not investigated forther.
`Infrared spectroscopic analysis of glucagon formulations
`was used to evaluate the ellects of freeze-drying and storage
`on glucagon's secondary structure, by comparing spectra for
`the peptide in the dried formulations to that for the native
`peptide in aqueous solution. The main secondary structure
`of native glucagon is a mixture of a-helix and random coil
`(35,36). Correspondingly, the infrared spectrum for native
`glucagon in aqueous solution has main absorbance around
`
`Page 5
`
`

`
`Effects of Excipients on Glucagon Stability in Solid
`
`Fig. 3 RP-HPLC
`chromatograms of glucagon and
`its chemical degradation
`products. (a) Control sample; (b)
`expanded scale of a; (c) oxidized
`glucagon by hydrogen peroxide;
`(d) degraded in 5 mM sodium
`sodium phosphate buffer, pH 3.0,
`at 60°C for 5 days; (e) following
`incubation of dry powder (freeze(cid:173)
`dried in sodium phosphate buffer)
`at 60°C for 2 weeks (scale was
`expanded for improved visual
`resolution of peaks).
`
`GOO
`
`a
`
`glucagon
`/
`
`I
`\ l
`
`5
`
`10 15 20 25 30 35 40 45
`Retention tirre (rrin)
`
`50
`
`20
`
`e 15
`
`<ol'
`
`l\i r 0
`
`5
`
`10
`
`b
`
`3283
`
`glucagon
`
`/
`
`JV\______
`
`15 20 25 30 35 40 45 50
`Retention tirre (rrin)
`
`d
`
`glucagon
`/
`
`c
`
`/
`
`oxidiz.ed glucagon
`
`2SO
`
`e 200
`
`<ol' 1: 0
`~: e
`/IJ~
`f:uV •lAlL
`
`5
`
`10 15 20 25 30 35 40 45
`Retention tirre (rrin)
`
`50
`
`5
`
`10 15 20 25 30 35 40 45 50
`Retention tirre (rrin)
`
`5
`
`10 15 20 25 30 35 40 45 50
`Retention tirre (rrin)
`
`1657 cm- 1 (Fig. 5). The peak is broad, presumably due to
`high flexibility of backbone conformation.
`
`For comparison of the native peptide spectrum to that of
`folly denatured and aggregated glucagon, the peptide
`
`Table I ESl-MS Analysis of Glucagon Hydrolysis Products After Incubating at 5 mM Sodium Phosphate Buffer, pH 3.0 at 60°C for 2 days
`
`Degradation product
`
`Glucagon [ 1-9]
`His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp
`Glucagon [I 0-29]
`Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr
`
`Glucagon [ 1-1 5]
`His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp
`
`Glucagon [ 16-29]
`Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-T rp-Leu-Met-Asn-Thr
`
`Glucagon [1-21]
`His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp
`
`Glucagon [22-29]
`Phe-Val-Gln-Trp-Leu-Met-Asn-Thr
`
`ESl-MS (m/z)
`
`Calculated
`
`[M +Ht =978.4
`[M + 22+=490.2
`[M + 2 Hf+= 1261 .6
`[M+3 H]3+=841.4
`[M+4 H]4+=631.3
`[M+2 H]2+=874.9
`[M+3 H]3+=583.6
`[M+2 H]2+=876.4
`[M+3 Hf'=584.6
`[M+3 H]3+=821.7
`[M + 4H]4+ =616.5
`[M +Ht= 1038.5
`[M + 2H]2+=5 I 9.8
`
`Observed
`
`[M + Ht=978.4
`[M + 2 H]2+ =490.2
`[M+2 Hf'=1261.6
`[M + 3 H]3+=84 I .4
`[M +4 H]4+=63 I .3
`[M + 2 H]2+=874.9
`[M+3 H]3+=583.6
`[M+2 H]2+=876.4
`[M+3 H]3'=584.6
`[M+3 H]3+=821.7
`[M + 4H]4+ =616.5
`[M+Ht= 1038.5
`[M + 2Hf+=5 I 9.8
`
`if:! Springer
`
`Page6
`
`

`
`3284
`
`g2.o
`Cl
`Ill u
`::I
`(5
`gi 1.5
`·2
`'iii
`E
`Cl)
`0::
`~ 1.0
`Cl
`0
`....I
`
`0
`
`50
`
`150
`100
`Time (min)
`
`200
`
`250
`
`Fig. 4 Oxidative degradation of glucagon with 0.6% H102 at 4°C. The
`% remaining glucagon was determined from chromatograms obtained
`by RP-HPLC.
`
`solution was incubated in a boiling water bath for 20 min and
`then freeze-dried. It ha~ been reported that upon fibrillation,
`the main secondary structure of glucagon is converted into
`intermolecular P-sheet (3 7 ,38). In the IR spectrum of boiled
`and freeze-dried glucagon the peak intensity around
`165 7 cm - I was greatly decreased and a new peak around
`1630 cm- 1 was observed (Fig. 5), suggesting intermolecular P(cid:173)
`sheet was formed between glucagon molecules during
`heating-induced aggregation.
`For analysis of the freeze-dried formulations, first the
`infrared spectra of glucagon formulations freeze-dried in
`glycine hydrochloride buffer were studied (Figs. Ga and
`7a). The dominant spectral feature affected by formulation
`was the intensity or the band at l G5 7 cm - I. Therefore this
`parameter was used to compare results for the difforent
`formulations (Fig. 7). Compared to the spectrum of the
`native, aqueous peptide, the band intensity was only slightly
`decreased in the spectrum for the peptide freeze-dried in
`bufler alone. There was also no increase o!' absorbance
`around 1630 cm- 1-a strong band in the spectrum of
`denatured/aggregated glucagon-suggesting no significant
`amount of intermolecular P-sheet was formed during freeze(cid:173)
`drying of glucagon in glycine buffer.
`The presence of polysorbate 20, HES or P-CD alone in
`the formulation did not result in substantial difference in the
`
`Fang et al.
`
`peak intensity at 1657 cm-1 compared to that for the peptide
`freeze-dried in buffer alone (Figs. Ga and 7a). In contrast,
`freeze-drying in formulations containing trehalose resulted
`in a substantial increase in band intensity due to narrowing
`of the band. This result is presumably because of an increase
`in the structural compaction of the peptide and a more
`narrow distribution of conformational substates than that
`present in aqueous solution. The mixture of trehalose and
`HES also preserved tl1e a-helix structure of glucagon, but
`was less effective compared with the formulation in which
`trehalose was the only excipient.
`Trehalose eflectively preserved the secondary structure of
`glucagon probably by hydrogen bonding to the peptide in
`place of water during drying, as has been observed with
`many proteins (G, 7). HES or P-CD probably could not form
`effective hydrogen-bonds with dried glucagon and, there(cid:173)
`fore, did not have a protective effect on the secondary
`structure of glucagon.
`The effects of incubation (G0°C for 2 weeks) of the freeze(cid:173)
`dried formulations prepared in glycine bufler on glucagon's
`secondary structure were also evaluated (Figs. Gb and 7b).
`Interestingly, the a-helix peak intensity for most formula(cid:173)
`tions was increased following incubation, suggesting that the
`secondary structure of glucagon was more ordered. The
`cause of this effect is not known.
`The eflects of excipients in sodium phosphate buffer were
`similar to those seen in glycine hydrochloride buffer (Fig. 7),
`with the greatest intensity of the l G5 7 cm - I band noted in
`freeze-dried formulations containing trehalose. Also, the
`peak intensities in the glucagon spectra were increased by
`incubation of the freeze-dried formulations at 60°C for
`2 week~.
`With sodium citrate buffer the eflects of excipients also
`were similar to those seen in glycine bu!Ier (Fig. 7a), with the
`greatest intensity of the 165 7 cm - I band noted in freeze(cid:173)
`dried formulations containing trehalose (Fig. 7a). However,
`unlike the results noted in formulations in glycine and sodi(cid:173)
`um phosphate buffers in which the presence of P-CD did not
`change the secondary structure compared to the formula(cid:173)
`tions without any excipient, there was increased band
`
`Table II MS-MS Analysis of Glu(cid:173)
`cagon and Oxidized Glucagon
`
`Parent species charge0
`
`Glucagonb
`
`Oxidized glucagonb
`
`5
`
`4
`
`3
`2
`
`3 Parent species with 2-5 charges
`were chosen for fragmentation.
`bThe main fragments were shown
`with charge state. The data sug(cid:173)
`gested the oxidation is in Met27
`residue
`
`~Springer
`
`365.2 (+I): y3 ion Met-Asn-Thr
`246.0 ( + I): Met-Asn
`780.4 (+4): b ion ofGlu(l-26)
`813.1 ( +4): b ion of Glu(l-27)
`365.2 ( + I): y3 ion Met-Asn-Thr
`246.0 ( + I): Met-Asn
`84 i .9 ( +4): b ion of Glu(l-28)
`876.5 ( +2): y ion of Glu(l6-29)
`1261. I (+2): y ion of Glu(I0-29)
`
`381.1 (+I) y3 ion of Met(O)-Asn-Thr
`262. I ( + I): Met(O)-Asn
`780.4 (+4): b ion ofGlu(l-26)
`817.2 ( +4): b ion of [O]Glu(l-27)
`381.1 (+I): y3 ion of Met(O)-Asn-Thr
`262. I ( + I): Met(O)-Asn
`845.9 ( +4): b ion of [O]Glu(l-28)
`884. 4 ( + 2): y ion of [O]Glu( 16-29)
`1269.6 ( + 2): y ion of [O]Glu(I 0-29)
`
`Page 7
`
`

`
`Effects of Excipients on Glucagon Stability in Solid
`
`3285
`
`Table Ill Composition and Tg (0C) of Freeze-Dried Glucagon Formulations
`
`Excipient
`
`Buffer0
`
`None
`
`PS 20
`
`Trehalose
`
`HES
`
`Tre-HES
`
`13-CD
`
`Glycine
`
`Glucagon: 90%
`Glycine: I 0%
`Glucagon: 89%
`Glycine: 9%
`PS 20: 2%
`Glucagon: 32%
`Glycine: 4%
`Tre: 64%
`Glucagon: 32%
`Glycine: 4%
`
`HES: 64%
`Glucagon: 32%
`Glycine: 4%
`Tre: 32%
`HES: 32%
`Glucagon: 32%
`Glycine: 4%
`
`13-CD: 64%
`
`133.1 ± 1.2 (3)
`
`140.0(1)
`
`105.3 ±0.6
`
`207.7± 1.8
`
`124.1 ±6.4
`
`218.2± 11.3
`
`Phosphate
`
`Glucagon: 90%
`Phosphate: I 0%
`Glucagon: 89%
`Phosphate: 9%
`PS 20: 2%
`Glucagon: 32%
`Phosphate: 4%
`Tre: 64%
`Glucagon: 32%
`Phosphate: 4%
`HES: 64%
`Glucagon: 32%
`Phosphate: 4%
`Tre: 32%
`HES: 32%
`Glucagon: 32%
`Phosphate: 4%
`
`13-CD: 64%
`
`140.1 ±0.7
`
`134.8±0.8
`
`11