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Deamidation, Acylation and Proteolysis of a Model Peptide in PLGA Films ⁎ M.L

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Deamidation, Acylation and Proteolysis of a Model Peptide in PLGA Films ⁎ M.L Journal of Controlled Release 112 (2006) 111–119 www.elsevier.com/locate/jconrel Deamidation, acylation and proteolysis of a model peptide in PLGA films ⁎ M.L. Houchin a, K. Heppert b, E.M. Topp a, a Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave., Lawrence, KS 66047, United States b Higuchi Biosciences Centers, The University of Kansas, Lawrence, KS, United States Received 8 December 2005; accepted 30 January 2006 Available online 9 March 2006 Abstract The relative rates of deamidation, acylation and proteolysis (i.e. amide bond cleavage) were determined for a model peptide (VYPNGA) in poly (DL-lactide-co-glycolide) films. Films were stored at 70°C and either 95%, 75%, 60%, 45%, 28%, or ∼0% relative humidity and at 37°C and 95% relative humidity. Peptide degradation products were identified by ESI+MS/MS and quantitated by LC/MS/MS. Extensive overlap of degradation mechanisms occurred, producing a complex mixture of products. Acylation was the dominant peptide degradation reaction (10–20% of total peptide) at early stages of PLGA hydrolysis and at intermediate relative humidity (60–45% RH). Deamidation and proteolysis were dominant (25–50% and 20–40% of total peptide, respectively) at later stages and at high relative humidity (95–75% RH). Understanding the relative rates of each peptide degradation reaction will allow for improved design of PLGA formulations that preserve the stability of peptide and protein drugs. © 2006 Elsevier B.V. All rights reserved. Keywords: PLGA; Deamidation; Acylation; Proteolysis; Peptide stability 1. Introduction bonds produces lactic and glycolic acid, which are easily metabolized. Polymer degradation and release rates can be Peptide and protein drugs have had the highest rate of sales tailored by altering the lactide to glycolide ratios and the growth of any drug class in the last decade and more than forty polymer molecular weight [4,5]. These properties have made new protein drugs have entered the market in the last twenty PLGA an attractive vehicle for protein and peptide delivery, years [1,2]. This rate of growth is expected to continue, with the leading to extensive research into PLGA encapsulation of number of peptide active pharmaceutical ingredients (APIs) protein drugs in a wide variety of delivery systems [5–12]. projected to increase at nearly double the rate of traditional, Despite these advantages, incorporation of protein drugs in small molecule APIs [2]. Parenteral routes of administration, PLGA often results in incomplete release from controlled including polymeric controlled release devices, provide an release devices, as well as physical and chemical degradation of attractive means of delivering intact protein drug by circum- the drug [5,10,13]. These instabilities have been attributed to the venting degradation in the G.I. tract and first pass metabolism production of free carboxylate groups upon the hydrolysis of by the liver [3]. Poly (lactide-co-glycolide) (PLGA), one of the PLGA, producing a reduced pH microclimate within the device few polymers approved by the FDA, has many desirable [13,14]. This acidic environment accelerates acid-catalyzed properties for protein controlled release. PLGA, a polyester, is degradation reactions such as deamidation and the hydrolysis of both biodegradable and biocompatible. Cleavage of the ester peptide bonds [15,16]. Physical and chemical interactions between PLGA and incorporated proteins have also been proposed as contributors to incomplete drug release [17–19]. Reaction of protein primary amines with ester or carboxylate ⁎ Corresponding author. Tel.: +1 785 864 4820; fax: +1 785 864 5736. groups via acylation has been identified as a specific source of E-mail address: [email protected] (E.M. Topp). these protein–polymer interactions [20–22]. 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.01.018 112 M.L. Houchin et al. / Journal of Controlled Release 112 (2006) 111–119 Attempts have been made to control pH within PLGA 2.3. Sample Analysis formulations by including buffers and basic salts [5,23,24], but these techniques may not prevent degradation reactions that are 2.3.1. ESI MS/MS both acid and base labile, such as deamidation. Other additives Product identification was accomplished by precursor and and peptide modifications have been implemented to prevent product ion scans of the mass of the VYPNGA and its known physical interactions between the PLGA and encapsulated degradation product masses. Prior to MS/MS analysis, the protein drugs, with moderate success [8,9,18,25,26]. sample films were dissolved in 0.5mL acetonitrile. To remove The studies reported here address the chemical stability of high molecular weight polymer fragments, 0.4mL aliquots were a model hexapeptide (VYPNGA) in PLGA films during centrifuged for 30min using Ultrafree-MC 5K NMWL filters storage. The peptide is subject to deamidation at the Asn (N) prior to injection (Millipore, Billerica, MA). The spectra were residue in acidic solution and in rubbery and glassy acquired using positive mode electrospray ionization (ESI+) amorphous solids [27–31]. Our studies show that cleavage tandem mass spectroscopy on a Micromass Quatro Micro Mass of the peptide bond and acylation also occur, and that the Spectrometer (Manchester, UK) using Mass Lynx software appearance of the products of the three reactions (deamida- (version 4.0). The sample was infused at a flow rate of tion, acylation, peptide bond hydrolysis) depends on time and 20 μL/min, with a capillary voltage of 3.5kV, a source storage conditions. temperature of 150°C and a cone voltage of 20V. The MS/ MS spectrum of ESI-MS ions was produced by collision- 2. Materials and methods induced decomposition (CID), using argon as the collision gas with a collision energy of 45eV. Both precursor and product 2.1. Materials scans were performed at an average rate of 1sec per 100 m/z. Val–Tyr–Pro–Asn–Gly–Ala (VYPNGA) (Asn-hexapep- 2.3.2. LC/MS/MS tide) was synthesized by American Peptide Company, Inc. Chromatographic separations were performed using a Waters (Sunnyvale, CA). All amino acids, except Gly, were in the L- 2690 HPLC system (Milford, MA) with an Econosphere C18 configuration. Peptide purity was determined by RP-HPLC to reversed-phase column (4.6×250mm, 5-μm particle size; be N95.0%. 50/50 Poly (DL-lactide-co-glycolide) (PLGA) was Alltech Associates, Inc., Deerfield, IL). The column was purchased from Absorbable Polymers International (Pelham, maintained at 25°C and the autosampler at 15°C. 50μL AL). The inherent viscosity of PLGA was 0.58dL/g and the volumes were injected. The total flow rate was 1.0mL/min. molecular weight was 75.4kDa. Acetonitrile, acetone, and Mobile phase A consisted of 0.05M ammonium acetate, buffer salts were purchased from Fisher Scientific (Fair Lawn, 1% (v/v) acetonitrile and 0.1% (v/v) formic acid. Mobile NJ). Ammonium acetate, formic acid, and trifluoroacetic acid phase B consisted of 0.05M ammonium acetate, 30% (v/v) (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). All acetonitrile, and 0.1% (v/v) formic acid. Both mobile phases water was deionized and purified using a Millipore MILLI-Q™ were filtered using 0.45μm nylon filters (GE Osmonics, Inc., water system. Minnetonka, MN) and sonicated prior to use. The gradient used for separation was 100% A from 0 to 5min, followed by a linear 2.2. Film preparation and storage gradient that increased the concentration of B from 0% to 100% B from 5 to 20min. The concentration of B was maintained for Films containing VYPNGA in PLGA were prepared by 1min, then returned from 100% to 0% B over the next 5min. dissolving 100mg/mL polymer and 300μg/mL peptide in The column was allowed to reequilibrate for 10min prior to the acetone and sonicating for 30min to enhance dissolution. A next sample injection. The HPLC elution stream was split, 0.5mL aliquot of the solution was then pipetted onto sending 0.35μL/min to the mass spectrometer and the rest to 22×22mm glass coverslips and allowed to dry for approxi- waste. mately 18h. Accelerated degradation studies were conducted at Detection was performed using ESI+tandem mass spectros- 70°C and either 95%, 75%, 60%, 45%, 28%, or ∼0% relative copy. Ionization parameters were as described above, with the humidity (% RH) achieved using K2SO4, NaCl, KI, NaBr, exception of collision energy, which was reduced to 30eV. MgCl2 saturated salt solutions and anhydrous calcium sulfate, Multiple reaction monitoring (MRM) experiments were per- respectively. An additional study was conducted at 37°C and formed with a dwell time of 200ms per compound. The 95% RH. Triplicate samples were removed from storage at following transitions were monitored: VYPNGA (Asn-hexa- appropriate times based upon the expected degradation rate of peptide), m/z 620.3N358.2; VYPDGA (Asp-hexapeptide), m/z the parent peptide. Upon removal, films were dissolved in 2mL 621.3N359.2; VYPUGA (Asu-hexapeptide), m / z of a solution containing 25% (v/v) acetonitrile, 0.05M 603.3N341.1; VYPD (tetrapeptide), m/z 493.3N231.1; Lac- ammonium acetate, and 0.1% (v/v) TFA (pH=4.5). To remove toyl–VYPUGA (Lac–Asu), m/z 675.15N603.35; Lactoyl– any PLGA precipitate, 0.5mL aliquots were centrifuged for VYPDGA (Lac–Asp), m/z 693.3N621.3; Glycoloyl-VYPUGA 3min using Gelman Sciences Nanosep GHP 0.45μm filters (Gly–Asu), m/z 661.2N603.35; Dilactoyl–VYPUGA (Lac-2- (Pall Corp., East Hills, NY). Prior to analysis, 100μL aliquots of Asu), m/z 747.15N603.35; Glycine (G), m/z 76.0N30.0; the sample mixture were spiked with 10μL of a 200μL/mL Alanine (A), m/z 90.0N44.0; Glycine–Alanine (GA), m/z stock solution of internal standard, VYPAGA.
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