Pharm Res (2012) 29:3278 -3291
`DOI 10.1007/s 1 1095 -012- 0820 -7
`
`RESEARCH PAPËR -`
`
`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 E Carpenter
`
`Received: 15 December 2011 /Accepted: 22 June 2012 /Published online: 6,July 2012
`© Springer Science +Business Media, LLC 2012
`
`ABSTRACT
`Purpose To evaluate the effects of several buffers and exci-
`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-
`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
`stability
`states
`
`J. F. Carpenter (2)
`W. -J. Fang W Qi
`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 94801, USA
`
`Present Address:
`W. -J. Fang
`Zhejiang Hisun Pharmaceutical Inc.
`46 Waisha Rd, Jiaojiang District,
`Taizhou, Zhejiang 3 1 8000, China
`
`Springer
`
`ABBREVIATIONS
`Abbreviations used for amino acids follow the rules of the IUPAC-
`IUB Joint Commission of Biochemical Nomenclature (Eur. J. Bio-
`chem. 1984
`138, 9 -37)
`CD
`cyclodextrin
`differential scanning calorimetry
`DSC
`electrospray ionization -mass spectrometry
`ESI-MS
`hydroxylethyl starch
`HES
`Infrared
`IR
`polyethylstyrene
`polysorbate
`reversed -phase high -performance liquid
`chromatography
`size exclusion
`glass transition temperature
`retention time
`
`PES
`
`PS
`RP-HPLC
`
`SE
`Tg
`
`tR
`
`INTRODUCTION
`
`Peptides have become increasingly important as therapeutic
`products. Currently there are more than 60 approved pep-
`tide drugs, and an additional 130 peptide candidates are in
`clinical development (1). As is the case with protein thera-
`peutics, peptide drugs are susceptible to both chemical and
`physical degradation, and stabilization of these products is
`challenging (2 -5). One approach to achieving sufficient
`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-
`ing the freeze -drying process and long -term storage in dried
`formulations, as well as the mechanisms for such stabiliza-
`tion (6-8). For peptide drugs there is a more limited litera-
`ture on stabilization in freeze -dried formulations (9 -12).
`Therefore, the goal of the current study was to gain further
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`Effects of Excipients on Glucagon Stability in Solid
`
`3279
`
`insights into how the lessons learned from protein therapeu-
`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
`during 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-
`ducing sugars glucose and fructose,(13) which can chemical-
`ly degrade proteins or peptides via the Malliard reaction
`(13). For example, the Malliard reaction has been reported
`when glucagon was formulated with the reducing disaccha-
`ride lactose (14). Trehalose is more resistant to acidic hy-
`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 Tg (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 (6,7,17).
`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 has been
`attributed to several different mechanisms including compet-
`ing with protein molecules for interfaces, increasing thermo-
`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-
`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-
`lations are not well understood. Among therapeutic pepti-
`des, insulin has been most studied in dried formulations.
`Although 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
`
`study, the degradation rate was dependent on the pH of
`aqueous solution prior to freeze- drying as well as the water
`content of the freeze -dried cake (9,10). Covalent dimeriza-
`tion of human insulin was substantially decreased by incor-
`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 (13- relaxation) and lower degradation
`rates (i.e. deamidation and dimerization) than insulin freeze -
`dried with dextran (12). The authors speculated [3-relaxation
`of insulin was reduced because of hydrogen bonding with
`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 [3- 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 the water moisture con-
`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 of insulin -
`induced hypoglycemia (23). Glucagon is known for its pro-
`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 (3 -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-
`cagon was purchased from the American Peptide Company
`(Sunnyvale, CA). [3-CD was purchased from Cyclodextrin
`Technologies Development, Inc. (High Springs, FL). Tre-
`halose dihydrate was purchased from J.T.Baker (Philips-
`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
`
`Page 2
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`

`
`3280
`
`Fang et al.
`
`His -Ser- Gln -Gly- Thr -Phe -Thr- Ser- Asp9 -Tyr- Ser- Lys -Tyr- Leu -Asp' 5- 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
`(0.45 pm) were purchased from Whatman (Maidstone, Eng-
`land). Polyethylstyrene (PES) membrane filters (0.2 um)
`were purchased from Millipore (Billerica, MA). 3 -ml lyoph-
`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 (Milwaukee,
`
`Acidic Degradation of Glucagon in Aqueous Solution
`
`Glucagon (0.5 mg /ml) was dissolved in 5 mM sodium phos-
`phate buffer (pH =3.0) and incubated for 5 days at 60 °C.
`Aliquots (0.5 mL) of the solution were removed at various
`times and centrifuged at 14,500 rpm for 10 min. The
`supernatant was analyzed by reversed -phase high -
`performance liquid chromatography (RP -HPLC) as de-
`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-
`cagon (1 mg /ml) was oxidized by incubation in 0.6 "/o hy-
`drogen peroxide (1-1202), 20% acetonitrile and 80% water
`with 10 mM HC1 at 4 °C for 210 min. Aliquots were re-
`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).
`
`.
`
`ESI -MS and MS -MS Analysis of Glucagon
`and its Degradation Products
`
`temperature. For MS -MS experiments on glucagon and
`oxidized glucagon, various m/z components were selected
`and fragmented with suitable collision energy to have rea-
`sonable amount of peaks following fragmentation.
`
`Sample Preparation for Freeze -Drying
`
`Glucagon (10 mg /mL) was dissolved in three different buf-
`fers (glycine hydrochloride, sodium phosphate, and sodium
`citrate buffers, 5 mM, pH 3.0). The solution was then mixed
`in a 1:1 (v /v) ratio with various excipient solutions (prepared
`at twice the desired concentration using corresponding buff-
`er) to obtain a final glucagon concentration of 5 mg /mL and
`the fmal desired excipient concentration of 0.01% for poly -
`sorbate 20 and 10 mg /mL for the carbohydrates. The
`solution was then filtered through 0.2 pm 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-
`tion vials (13 -mm ID) and freeze -dried in a FT'S Durastop
`freeze -drier (Stoneridge, NY). For freezing, samples were
`cooled to -40 °C at 2.5 °C /min and maintained at this tem-
`perature for 2 h. Then the shelf temperature was increased to -
`5°C at 2 °C /min and held for 2 h as an annealing step (29). The
`temperature was then decreased to -30 °C at 1.5 °C /min and
`the chamber pressure was reduced to 8 Pascal. These condi-
`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 10 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 products were analyzed with
`Following freeze -drying, the sample vials were incubated at
`an electrospray -triple quadrupole- time -of- flight mass spec-
`trometer (ESI- gTOF -MS) from Applied Biosystems (PE
`60 °C and analyzed after 2 weeks. Triplicate sample vials for
`SCIEX /ABI API QSTAR Pulsar i Hybrid LC /MS/
`each formulation were incubated. Control sample vials
`(equivalent to time zero of incubation) were stored at -80°
`MSESL). Mass Spectra were acquired by scanning a mass -
`to- charge ratios (m /z) range from 100 to 2000. Eluates
`C until analysis. Following storage, formulations were first
`rehydrated to 5 mg /mL with water and then diluted to
`(1 mg /m1 glucagon, 50 µl) were injected into the mass
`1 mg /mL glucagon with the corresponding buffer. An ali-
`spectrometer at a flow rate of 5 µl /min. Spray voltage was
`quot of the rehydrated sample (200 µL) was analyzed for
`set at 4500 V, and capillary temperature was set at ambient
`
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`
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`
`Effects of Excipients on Glucagon Stability in Solid
`
`3281
`
`turbidity (see below). Then, the solution was centrifuged
`(14,500 rpm x 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)(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
`The glass transition temperature (T) of dried formulations
`Chromatography (RP -HPLC)
`was determined using a Perkin -Elmer Pyris -1 DSC (Nor-
`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: 28.45 J/
`g). In a dry 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 Tg, the sam-
`ples were first heated to above Tg to remove thermal history,
`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, which was determined as the midpoint of the
`transition.
`
`Chemical degradation of glucagon formulations was quan-
`tified by RP -HPLC, using an Agilent 1100 HPLC system
`(Agilent Technologies, Inc., Waldbronn, Karlsruhe, Ger-
`many) with a Thermo Biobasic C8 column (5 gm, 250 x
`4.0 mm ID, Waltham, MA) and a Restek VWD G1314Á
`detector (Bellefonte, PA). The mobile phase was 73%
`phosphate -cysteine buffer (pH 2.6) with 27% acetonitrile
`(USP 30 Official Monograph for Glucagon, pp 2230-
`2231, 2007). Before use, mobile phase was filtered with
`0.45 um Nylon membrane filters and degassed. The flow
`rate for the analysis was 1 ml /min, and elution was moni-
`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 -
`drying and storage.
`
`Infrared (IR) Spectroscopy
`
`Infrared spectra of glucagon in aqueous solution and in
`freeze -dried formulations were collected at room tempera-
`ture using a Bomern Prota spectrometer (Quebec, Canada)
`purged with dry 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 um 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-1. 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-I) and overlaid (30).
`
`Size Exclusion -HPLC (SE -HPLC)
`
`Aggregation of glucagon in different formulations was de-
`termined with SE -HPLC, using an Agilent 1100 HPLC
`equipped with a TSK G2000SW,t1 gel filtration column
`(5 gm, 300 x 7.8 mm ID) and a Restek DAD G1315A
`detector. The mobile phase was 3.2 mM HCI, 100 m1VI
`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-
`lated based on the monomer area of the formulation sam-
`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 (OD405)
`
`The presence of aggregates in rehydrated formulations
`(1 mg /mI, 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
`
`Page 4
`
`

`
`3282
`
`Fang et al.
`
`peaks with similar tR and almost identical areas were ob-
`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 += 1167.1, [M +4 H]4 += 875.6,
`[M +5 H]' += 700.7), suggesting one residue of glucagon was
`oxidized. NIS /1\4S analysis (Table II) of the oxidation prod-
`ucts showed the Met residue at position 27 was oxidized.
`The two peaks in the RP -HPLC chromatograms were prob-
`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 butlers
`(glycine hydrochloride, sodium phosphate and sodium cit-
`rate buffers, 5 mM, pH 3.0), polysorbate 20 and three
`carbohydrate excipients, trehalose, HES and [3-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 Tg) were also characterized for each freeze -dried
`formulation.
`The Tg of the freeze -dried formulations was determined
`by DSC (Table III). When there was no excipient, the Tg of
`dry powders was between 122 °C and 140 °C, depending on
`the buffer used in formulation (Table III). As expected, the
`presence of polysorbate 20 (0.01 %w /v) did not have much
`effect on Tg.
`For formulations with glass forming carbohydrates, the
`Tg was highest fbr HES and (3 -CD, followed by the mixture
`of trehalose and HES, and then trehalose alone (Table III).
`These values were consistent with the Tg of the
`corresponding pure carbohydrates and their mass ratios in
`the formulations. The buffer also had a substantial effect on
`Tg, especially in the cases where HES and 13 -CD were used
`as excipients (Table III). The cause(s) of the buffer effect was
`not investigated further.
`Infrared spectroscopic analysis of glucagon formulations
`was used to evaluate the effects of freeze -drying and storage
`on glucagon's secondary structure, by comparing spectra Íòr
`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
`
`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-
`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 function of incubation time.
`The commercial glucagon we used had some impurities
`prior to incubation (about 8 -9 %, see Fig. 3a and its en-
`larged version Fig. 3b). During incubation, glucagon under-
`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
`(tit) around 7, 14, 19 and 29 min, respectively) and several
`smaller peaks (i.e. tit around 33, 37, 40, and 42 min) were
`observed. The main peaks were probably due to Asp cleav-
`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
`affinity of glucagon to its receptor (33), the potency the
`oxidized peptide in stimulating glucose production in isolat-
`ed rat adipocytes and hepatocytes is decreased (34). There-
`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
`
`Em
`
`o
`
`2
`
`4
`
`6
`
`Time (days)
`
`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
`
`

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`

`
`Effects of Excipients on Glucagon Stability in Solid
`
`3285
`
`Table Ill Composition and Tg ( °C) of Freeze -Dried Glucagon Formulations
`
`Excipient
`
`None
`
`PS 20
`
`Trehalose
`
`HES
`
`Tre -HES
`
`ß-CD
`
`Buffer°
`
`Glycine
`
`Glucagon: 90%
`Glycine: 10%
`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%
`(3 -CD: 64%
`
`133.1 ± 1.2 (3)
`
`140.0 (I)
`
`105.3 ± 0.6
`
`207.7 ± 1.8
`
`124,1 ±6.4
`
`218.2± 11.3
`
`Phosphate
`
`Glucagon: 90%
`Phosphate: 10%
`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%
`(3 -CD: 64%
`
`140.1 ±0.7
`
`134.8 ±0.8
`
`110.0±0.6
`
`154.0 ± 0.9
`
`131.3 (I)
`
`152.8 ±-1.7
`
`Citrate
`
`Glucagon: 83%
`Citrate: 17%
`
`Glucagon: 82%
`Citrate: 16%
`PS 20: 2%
`
`Glucagon: 31%
`Citrate. 6%
`Tre: 63%
`
`Glucagon: 31%
`Citrate: 6%
`HES: 63%
`
`Glucagon: 31%
`Citrate: 6%
`Tre: 32%
`HES: 32%
`
`Glucagon: 31%
`Citrate: 6%
`ß -CD: 63%
`
`121.5±0.4
`
`119.9 ± 0.8
`
`110.4±0.3
`
`173.2 ± 0.4
`
`119.8 ± 0.9
`
`136.5± 1.6
`
`°The values are the mean ±SD from two independent sample replicates unless otherwise specified
`
`determined by SE -HPLC. Although 3 -CD has relatively low
`solubility in water (18.5 mg /ml at room temperature(39)), the
`
`a
`
`o.6oat` ___....--._..._._..._......__'y
`
`..r
`
`1
`
`.gr -0.02
`
`°
`
`.04
`
`1700
`
`1660
`1640
`1620
`1660
`Wavenumber (cm'')
`
`b
`
`.00
`
`á .0.02
`
`.0.04
`
`1700
`
`ï
`
`1680
`
`1660
`
`1620
`Wavenumber (cm4)
`
`1640
`
`- Native
`-- None
`_ PS 20
`Trehalose
`HES
`--- Tre-HES
`ft-co
`1600
`
`- Native
`None
`- PS 20
`-- Trehalose
`HES
`°- Tre-HES
`f1-CB
`
`1600
`
`Fig. 6 Area -normalized second -derivative infrared spectra of glucagon
`when freeze -dried in glycine hydrochloride buffer in the absence of
`additives (None) or in the presence of polysorbate 20 (PS 20),
`I/I mixture of trehalose and HES (Tre -HES), and
`trehalose, HES,
`p -CD. The infrared spectrum of the native glucagon in aqueous
`solution was also shown for comparison. (a) After freeze -drying; (b)
`after incubation at 60 °C for 2 weeks.
`
`Springer
`
`Page 8
`
`intensity (at 1657 cm -1) in spectrum of glucagon in the 3-
`CD formulat