Method for Characterization of PEGylated Bioproducts in Biological

Sep 25, 2013 - PEGylation of peptides and proteins has been widely used to enhance stability and reduce immunogenicity of biotherapeutics. Characteriz...
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Method for Characterization of PEGylated Bioproducts in Biological Matrixes Qingyuan Liu, Michael R. De Felippis, and Lihua Huang* Bioproduct Research & Development, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States S Supporting Information *

ABSTRACT: PEGylation of peptides and proteins has been widely used to enhance stability and reduce immunogenicity of biotherapeutics. Characterizing the degradation of these PEGylated products in biological fluids can yield essential information to support pharmacokinetic evaluations and provide clues about their in vivo properties useful for further molecular optimization. In this paper, we describe a novel and uncomplicated approach to characterize PEGylated peptides or proteins and their related degradation products in biological matrixes. The method involves direct liquid chromatography/mass spectrometry (LC/MS) analysis of animal sera containing low nanograms to low micrograms per milliliter of PEGylated product with or without an acetonitrile precipitation sample treatment. Applying the methodology to analyze the model PEGylated peptides, 20K PEGylated-Pancreatic Polypeptide analogue (PPA) and 20K PEGylated-glucagon, we elucidated the decomposition pathways occurring in animal sera. The data provided direct evidence of cleavages within the peptide backbone. The identified degradation products were unambiguously confirmed by tandem mass spectrometry with high-energy C-trap dissociation (HCD) analysis, followed with in-source fragmentation. Additional spiking studies demonstrated nearly full recovery of PEGylated products, linear detection when the spiked concentration of PEGylated product was ≤1000 ng/mL, and a low ng/ mL limit of quantitation (LOQ).

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are 10 PEGylated products on the market which involve conjugation with proteins, oligonucleotides, and antibody fragments.11,12 Numerous studies have demonstrated that PEGylation significantly enhances the pharmacological properties of conjugates through the increase of solubility, improvement of chemical and physical stability, reduction of immunogenicity and tissue uptake, and extension of circulating lifespan, mostly due to the size-increasing and shielding effects of PEG moieties. In addition, toxicological studies of PEGylated proteins have not revealed any significant PEG-related toxicity.13−17 Several recently published reports describe methodologies for characterization of PEGylated products.18−20 However, to characterize PEGylated biotherapeutics in biological matrixes and clearly relate the information to the pharmacokinetic profile, highly sensitive, reproducible, and specific analytical methodology is needed that can ideally interrogate both the protein/peptide and PEG components of the conjugate. Immunoassays, bioassays, and protein radiolabeling are the most commonly employed bioanalytical methods for pharmacokinetic studies.12,21,22 Although radioisotope labeling has been successfully utilized for pharmacokinetic study of PEG

roteins and peptides have been broadly utilized by the pharmaceutical industry as effective therapeutic agents for many diseases, and successes in commercialization have been aided in part by the rapid developments in recombinant DNA and solid-phase synthetic technologies over the past decades.1,2 Despite these advances in production capability, protein and peptide pharmaceuticals still suffer from significant limitations, including low bioavailability, rapid degradation and clearance, low solubility at physiological pH, and potential immunogenicity. These issues coupled with the need to deliver these products by injection have generally hindered development and commercialization of peptides and proteins. Many technologies have been introduced to overcome these obstacles and modulate the pharmacological properties of potential protein and peptide therapeutics. These strategies mainly involve amino acid manipulation, molecular derivatization including fusion to albumins and conjugation with nontoxic polymers, and drugdelivery systems such as application of liposomes, microspheres, and nanoparticles.3−8 PEGylation, a well-established molecular derivatization strategy, introduces the water-soluble polymer poly(ethylene glycol) through covalent attachment to the target molecule. PEGylation has become one of the most attractive pharmaceutical research areas since Frank Davis and Abraham Abuchowski demonstrated the feasibility of protein pegylation in the late 1970s.9,10 The first PEGylated protein Adagen (Enzon) was approved by the FDA in 1990. At present, there © XXXX American Chemical Society

Received: June 18, 2013 Accepted: September 13, 2013

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combining 100 μL of the spiked solution with 100 μL of 0.5% TFA in acetonitrile, mixing well, and centrifuging at 15 000g for 2 min. The resulting supernatant was then removed for LC/MS analysis. Spike and Recovery Samples of 20K PEG Insulin Analogue and 20K PEG. Stock solutions of 20K PEG-Insulin Lispro28 and 20K PEG were separately spiked into monkey serum and a control solution consisting of 2 mg/mL BSA in 135 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4 (1× PBS) to yield the intended concentrations of 5 ng/mL to 10 000 ng/mL. The spiked serum and control samples were mixed with 0.5% TFA in acetonitrile at a ratio of 1:1 (v/v) and then centrifuged at 15 000 rpm for 2 min. Supernatants were collected and analyzed using LC/MS. PEG Stability Samples in Biological Matrixes. Stock solutions of 20K PEG-PPA (1-WAPLEPVYPGECATPEQL ARYAADLRRYINALTRPRY-37) or 20K PEG-glucagon were diluted and spiked into mouse (or monkey) serum (or plasma) at a final concentration of 1 μg/mL (based on peptide content). After incubation at 37 °C for 0, 2, 6, and 24 h, the samples were mixed with 100% of acetonitrile at a ratio of 1:1 (v/v), centrifuged at 15 000g for 10 min, and the supernatants were collected for LC/MS and LC/MS/MS analysis. Ultra Performance Liquid Chromatography/Mass Spectrometry (UPLC/MS) Analyses. Characterization of 15K and 20K PEG 6K Peptide, 20K PEG-Lys, and 15, 20, 30, and 40K PEG spiked into monkey serum or BSA solution was performed using a Waters (Milford, MA) SYNAPT G2-S mass spectrometer interfaced with a Waters Acquity UPLC system. Samples were separated on a reversed-phase, polymeric column (PLRP-S, 1.0 mm × 50 mm, 1000 Å, 5 μm particles, Agilent) using 0.01% trifluoroacetic acid (TFA) in water as mobile phase A and 0.01% TFA in acetonitrile as mobile phase B. The gradient program was as follows: 48% mobile phase B at a flow rate of 200 μL/min and hold for 1.5 min, flow rate reduced to 100 μL/min in 0.1 min, mobile B increased to 59% in 15.3 min, and mobile B increased to 100% at a flow rate of 600 μL/min in 0.2 min. Following a postcolumn addition of DEMA at 2.5 μL/ min of 0.5% DEMA in 50% acetonitrile aqueous solution, the eluted samples were introduced into the mass spectrometer through an electrospray ionization (ESI) source operating under the sensitivity mode and at a cone voltage of 130 V, a capillary voltage of 3.10 kV, a desolvation gas flow of 900 L/h, and a desolvation temperature of 550 °C. Depending on the HPLC system used and PEGylated product(s) being analyzed, the HPLC gradient may require adjustment to ensure the PEGylated product elutes in the low flow rate range. UPLC/MS/MS Analysis. Characterization of PEGylated peptides was performed using a Thermo (San Jose, CA) LTQ Velos Pro Orbitrap Elite mass spectrometer coupled with a Waters Acquity UPLC system. UPLC parameter settings were similar to those described above. The eluted PEG-peptides were analyzed using an ESI source operating at a spray voltage of 4.4 kV, a capillary temperature of 325 °C, and a sheath gas setting of 26 (arbitrary units). In-source fragmentation at a voltage of 60 V was applied to obtain the small PEG fragments attached to the peptides. For characterizing the peptides attached with small PEG fragments, the selected precursor ion was subjected to HCD fragmentation at a collision energy of 25 V and an activation time of 8 ms.

conjugates, the methodology suffers from low specificity for the parent compound, limited readily available radioisotopes, and lack of dual labeled probes for both moieties in the PEG conjugates.23 Enzyme-linked immunosorbent assay (ELISA) is also a widely used technique for pharmacokinetic study due to its high sensitivity.24 An anti-PEG ELISA was recently developed by Epitomics for PEG-conjugates detection; however, this PEG-specific ELISA only provides reliable results for multi-PEGylated conjugates and has low sensitivity for detecting trace amounts of mono-PEGylated conjugates in blood sera.12,25 The application of a protein-specific ELISA for metabolic profiling of PEGylated proteins is also hindered by the low sensitivity, because the shielding effect of PEGylation partially blocks the receptor binding sites. Furthermore, both radioisotope labeling and ELISA cannot generally provide information on the degradation pathways of PEGylated products in vivo. A combined analytical approach comprising immunoblotting and nuclear magnetic resonance (NMR) was published recently.26 The combined methodologies were used to simultaneously monitor the protein and PEG components of a 40K PEGylated-insulin incubated in rat serum and obtain evidence of conjugate dissociation. All of all these published approaches lack the ability to obtain detailed information on the degradation pathways of the individual protein/peptide and PEG components associated with PEGylated products, and no single analytical method provides sufficient, direct, and precise characterization data necessary to support pharmacokinetic studies. Here we describe a novel and uncomplicated approach to characterize PEGylated products in biological matrixes and demonstrate its utility by investigating the serum stability of model PEGylated peptides. The methodology provides direct observation of PEGylated peptide degradation occurring in serum samples using mass spectrometry analysis, and the structural information associated with degradation is confirmed through high-energy C-trap dissociation (HCD).

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EXPERIMENTAL SECTION Materials. All chemicals were reagent grade or higher purity and obtained from commercial sources. Chromatographic solvents were HPLC grade. The PEGylated peptides (see the Supporting Information) were produced at Eli Lilly and Company (Indianapolis, IN) or made according to previously published procedures.19,20,27 Free PEG samples were prepared by deactivating 15, 20, and 40K α-methoxy-ω-(4-nitrophenoxy carbonyl), polyoxyethylene (15, 20, and 40K mPEG-PNP), which were purchased from NOF America Corporation (Irvine, CA). Mouse and monkey sera were obtained from EquitechBio, Inc. (Kerrville, TX) and mouse plasma was from LAMPIRE Biological Laboratories, Inc. (Pipersville, PA). Diethylmethylamine (DEMA) was purchased from SigmaAldrich Co. (St. Louis, MO). Monkey Serum or BSA Solution Samples Spiked with PEGylated Products. A 930 μL aliquot of monkey serum or a solution of 2 mg/mL BSA in 135 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4 (1× PBS), was mixed with 10 μL of a 1 mg/mL solution of each PEGylated product (15K and 20K PEG 6K Peptide, 20K PEG-Lys, 15, 20, 30, and 40K PEG, see the Supporting Information for details). A 200 μL volume of each spiked solution and monkey serum alone was centrifuged at 15 000g for 2 min and the supernatant removed for LC/MS analysis. The monkey serum spiked with PEGylated products was also subjected to a precipitation procedure that involved B

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Figure 1. Total ion chromatograms obtained by LC/MS analysis following a postcolumn neutralization with DEMA. (a) Monkey serum spiked with 10 μg/mL each of 15K and 20K PEG insulin analogue (InsA); 20K PEG-Lys; and 15, 20, 30 and 40K PEG. The injection volume was 1 μL. (b) Same sample set as in part a but with injection of 2 μL of the supernatant obtained after mixing with 0.5% TFA in acetonitrile in a 1:1 ratio. (c.) Same sample set as in part a but 10 μg/mL of each PEG spiked into a solution of 2 mg/mL BSA in PBS. Injection volume was 1 μL. (d.) Injection of 1 μL of monkey serum.

Figure 2. Mass spectra for the samples described in Figure 1. (a) Monkey serum spiked with 10 μg/mL each of 15K and 20K PEG insulin analogue; 20K PEG-Lys; and 15, 20, 30, and 40K PEG. (b) Same sample set as in part a after mixing with 0.5% TFA in acetonitrile in a 1:1 ratio. (c) Same sample set as in part a but 10 μg/mL of each PEG spiked into a solution of 2 mg/mL BSA in PBS. (d) Monkey serum. The major charged ions, triple for 15K PEG; quadruple for 20K PEGylated Lys and 20K PEG; quintuple for 15 and 20K PEGylated insulin analogue and 30K PEG; and sextuple for 40K PEG are shown for each PEGylated product.

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RESULTS AND DISCUSSION

generally required to completely or partially remove interfering substances before mass spectrometry analysis. These procedures can be labor intensive, particularly when attempting to obtain intact mass information of large molecules present at low ng/mL levels. Our method circumvents complicated sample cleanup steps, allowing direct collection of intact molecular weight information of PEGylated products in biological fluids. Following removal of contaminating debris

LC/MS Method for Characterization of PEGylated Products in Biological Fluids. Biological fluids, such as serum or plasma, contain a multitude of peptides, proteins, lipid, DNA, and other molecular components that may complicate characterization of PEGylated products by mass spectrometry. To address this issue, a sample clean up step is C

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Figure 3. Plot of intensity of the exacted ion chromatogram peak of the top 15 quintuple-charged ions of 20K PEGylated-insulin analogue and the top 15 quadruple-charged ions of 20K PEG versus the concentration of 20K PEGylated products spiked into monkey serum or 2 mg/mL BSA solution.

proteins do not bind to the column resin if the samples are injected at more than 45% acetonitrile in the mobile phase. Second, lipids, fats, or hydrophobic low mass molecules in the plasma or serum will bind to the resin but they either need a much higher percentage of acetonitrile to elute from the column or their ions are in a lower m/z range than PEGylated products when analyzed following postcolumn addition of DEMA. While the plasma or serum samples containing PEGylated products could be directly injected for LC/MS analysis, it is preferable to mix the sample with acetonitrile in a ratio of 1:1 (v/v) to precipitate plasma or serum proteins before the injection to avoid potential clogging of the HPLC system. These spiking studies also provide insight into the range of pegylated products that can be evaluated with our methodology. As shown in Figure 1, the 20K PEGylated 6K Peptide eluted earlier than 20K PEGylated Lys, while 20K free PEG eluted last. This interesting elution profile whereby PEGylated products containing larger peptide or protein molecules conjugated to a constant PEG size have shorter retention times on reversed phase columns has been previously observed.31 On the basis of this published data and the results of the spiking studies reported here, our method is suitable for analyzing peptides and proteins conjugated with PEG in the size range of 15 to 40K. Since molecular design strategies typically employ PEGs of this size, our method is broadly applicable to most peptide or protein products. Further studies were conducted to assess method capability. The linearity over a range of concentrations was determined by evaluating the recovery of the PEG conjugates. Samples were prepared containing varying amounts of 20K PEG-6K Peptide and 20K PEG mixed with 2 mg/mL BSA solution or monkey serum. Standard curves were prepared by plotting the known concentrations of the PEGylated conjugates and their corresponding MS signal intensities as shown in Figure 3. The mass intensities were extracted from the top 15 most

by either centrifugation or treatment with acetonitrile and centrifugation, biological fluids containing PEGylated products are directly analyzed using RP-LC/MS with a postcolumn addition of DEMA. Samples are injected under mobile phase conditions employing a high level of acetonitrile (approximately 50%) to resolve the PEGylated products from biological components. Figure 1 shows the total ion chromatograms obtained for monkey serum or 2 mg/mL BSA solution spiked with 10 μg/ mL of 15, 20, 30, and 40K free PEG, 15 and 20K PEGylated 6K Peptide, and 20K PEGylated Lys. The control sample, containing only monkey serum (Figure 1d), showed one peak in the retention time window of the PEGylated products, while seven peaks were detected for the spiked monkey sera without (Figure 1a) or with (Figure 1b) acetonitrile precipitation prior to the injection. A similar elution profile was obtained for the PEGylated products spiked into PBS buffer containing BSA (Figure 1c). The peak detected in the control sample was also found in the spiked monkey sera and eluted close to the 15K PEGylated peptide peak. No specific ions at significant levels above baseline were detected (see Figure 2), and it was concluded that this peak does not interfere with the characterization and quantitation of the 15K PEGylated product. Plasma or serum contains hundreds of proteins with a broad dynamic range from 10 mg/mL albumin that can possibly interfere with the intact mass detection of PEGylated products.29,30 Despite these complexities, our methodology does not suffer from limitations with analyte detection. Two chemical principles make it possible for a simple reversed phase HPLC step to remove almost all serum proteins and capture the PEGylated products of interest for mass spectrometry analysis. First, PEGylated products are very soluble in water or in water−acetonitrile mixtures, but they are also very hydrophobic allowing favorable interactions with the reversed phase column resin. Most of the serum or plasma D

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Figure 4. Total ion chromatograms obtained by LC/MS analysis of the monkey serum stability samples spiked with 1 μg/mL 20K PEG-PPA at 0 (a), 2 (b), 6 (c), and 24 (d) h incubation and deconvoluted mass spectra obtained for PEGylated-PPA incubated in monkey serum after 0 h (e), 2 h (f), 6 h (g), and 24 h (h). Mass shifting was observed at 6 h and trended toward completion after 24 h incubation.

Table 1. Assignment of Peptide Moiety and the Number of Attached Ethylene Glycol Units for the Five Most Intense Peaks in 20K PEGylated PPA Control Sample and 24 h Serum Incubation Samplea 20K PEGylated-PEP A control sample observed MW 25 507 25 552 25 596 25 640 25 684 a

peptide fragments PPA PPA PPA PPA PPA

(1-37) (1-37) (1-37) (1-37) (1-37)

20K PEGylated-PPA 24 h serum sample

no. of oxyethylene

calculated MW

observed MW

478 479 480 481 482

25 507.4 25 551.5 25 595.6 25 639.6 25 683.7

25345 25389 25433 25478 25522

peptide fragments PPA PPA PPA PPA PPA

(1-36) (1-36) (1-36) (1-36) (1-36)

no. of oxyethylene

calculated MW

478 479 480 481 482

25 345.3 25 389.3 25 433.4 25 477.4 25 521.5

Intact PPA contains a C-terminal Tyr residue with a carboxamide group.

incubation for various times, aliquots were removed, mixed with acetonitrile at a final ratio of 1:1, centrifuged and the supernatant was directly subjected to LC/MS analysis with a postcolumn neutralization of diethylmethyamine (DEMA). Total ion chromatograms are shown in Figure 4a−d. A single peak was detected, but its retention time was slightly shifted with longer incubation times. A new series of ions was detected after 2 h incubation, and the original series of 20K PEG-PPA ions were almost completely eliminated after 24 h incubation according to mass spectra of the five charged ions corresponding to the 20K PEGylated-PPA peak (data not shown). Mass spectrum deconvolution was necessary to characterize the nature of the degradation. Figure 4f−h shows the deconvoluted spectra of 20K PEGylated-PPA after incubating in monkey serum for 0, 2, 6, and 24 h. The intensity of a new series of masses that highly suggest the generation of a new PEG-conjugate was observed after 2 h incubation and increased with the increasing incubation time. About half of the PEGylated-PPA was found to degrade after 6 h of incubation, and conversion of PEGylated PPA to the degradation product was almost complete after 24 h. The composition and molecular weight of the peptide moiety and the number of attached oxyethylene units for the most intense peaks in Figure 4f−h were calculated and listed in Table 1. The degradation pattern was identified by comparing and matching the number of attached oxyethylene units from the incubated serum sample with the control sample (0 h incubation). For the 0 h incubation serum sample, the most intense peaks were

intense mass ions corresponding to each concentration. By comparing the results obtained for samples spiked into 2 mg/ mL BSA solution, the recoveries of PEGylated product were determined to be nearly complete at the range of 1 μg/mL or lower in serum. The observed sample loss may be due to surface absorption and/or slight precipitation caused by the acetonitrile sample treatment procedure beyond this range of spike concentration. These experiments also demonstrated that the limit of quantitation (signal/noise ≥10) is approximately 10−20 ng/mL PEGylated product (data not shown). It should be noted that the exact limit of detection or quantitation is dependent upon the sensitivity of the mass spectrometer or HPLC conditions (e.g., increasing injection volume or using small diameter column) employed. To demonstrate application of our methodology, studies were conducted using two model peptides, PPA and glucagon, each pegylated with a 20 kDa PEG moiety. These conjugates were synthesized using two different types of PEGylation chemistries. PEGylated PPA was prepared through alkylation of the sulfhydryl group of a cysteine residue with 20K mPEGIAM. The PEGylated glucagon sample was synthesized by reacting 20K mPEG-PNP with the amino side chain of a lysine residue. Each pegylated product was characterized following incubation in serum to evaluate the effectiveness of the method for detecting specific structural changes. The results of these experiments are presented in the following sections. Structural Characterization of 20K PEGylated-PPA Degradation in Sera. The purified 20K PEGylated-PPA sample was spiked into mouse and monkey sera. After E

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Figure 5. (a) Mass spectrum of PEGylated-PPA following incubation in monkey serum. The attached truncated PEG fragments are generated after in-source fragmentation. (b) Tandem mass spectrum of the selected precursor ion at m/z of 1245.66.

Figure 6. Deconvoluted mass spectrum obtained for PEGylated-glucagon after 0 h (a), 2h (b), 6 h (c), and 24 h (d) incubation. The new mass distribution of polydispersed PEG-conjugate started after incubation for 6 h and completely formed at the 24 h incubation time.

the b1 to b9 ions and y1 to y16 ions, which exactly matched the theoretical mass of PPA (1-36) fragments (Figure 5b). Additionally, the mass shifts from y26 to y28, plus b13, supported that the PEGylation site is located at residue Cys12 of 20K PEGylated-PPA (1-36). Structural Characterization of 20K PEGylated-Glucagon Degradation in Plasma. 20K PEGylated-glucagon was spiked into mouse plasma and incubated for different times. Following treatment of the sample with acetonitrile to remove potential interfering substances, degraded 20K PEGylatedglucagon contained in the supernatant was directly analyzed by LC/MS. The deconvoluted spectra exhibited a new mass distribution at the lower mass range, suggesting the generation of a PEG-conjugate with a cleavage of the glucagon backbone (Figure 6). A total of 10 of the greatest intensity peaks of 20K PEGylated-glucagon were determined to contain intact glucagon (1-29) and 448 to 457 of oxyethylene units. For the spiked plasma samples, the composition of the most intense peaks was determined and compared to that of 20K PEGylatedglucagon (1-29). By matching the number of oxyethylene units,

calculated and assigned to PPA (1-37) and a series of polydispersed oxyethylene units (from 478 to 482). The assigned composition for the most intense peaks from the 24 h incubation serum sample perfectly matched the PPA (1-36) with the same number of oxyethylene unit polydispersity. The results clearly indicate a single C-terminal amino acid cleavage of the PPA backbone that occurred during the serum incubation. The degradation of 20K PEGylated-PPA in mouse serum was also investigated, and the results demonstrated the same degradation product (data not shown). To confirm the degradation product, LC/MS/MS associated with in-source fragmentation (ISF) was performed. The serum sample from the 24 h incubation was used since the complete degradation maximizes spectral intensity of the modified PEGpeptide and reduces the interference caused by the parent PEGpeptide. A series of quadruple-charged ions were observed after in-source fragmentation (Figure 5a). The quadruple-charged isotopic ion at m/z of 1245.66, which was calculated to consist of 16 oxyethylene units, was selected as the precursor ion for HCD MS/MS analysis. The tandem mass spectrum provided F

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ACKNOWLEDGMENTS The authors thank Dr. Jorge Alsina-Fernandez for providing the 20 kDa PEGylated-Pancreatic Polypeptide analogue.

the peaks were assigned to a fragment of glucagon (1-18). This assignment indicates that a cleavage reaction occurs at the carboxyl side of the Arg18 residue of 20K PEGylated-glucagon during incubation in plasma. The cleavage site identified was confirmed by a tandem MS associated with in-source fragmentation (data not shown). Previous reports indicate that glucagon can degrade in vivo by a number of pathways.32−35 One of the dominant degradation reactions generates a fully active fragment of glucagon (19-29) designated, miniglucagon, resulting from proteolysis by an endopeptidase enzyme.36−38 The product of 20K PEGglucagon degradation in plasma identified in our work is consistent with the previously proposed glucagon degradation pathway in vivo.

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CONCLUSION The ability to understand the in vivo degradation pathways of PEGylated biologics provides valuable information for optimizing the molecular properties of these potential therapeutic agents. However, it has been very challenging to study the metabolism of PEG conjugates in biological fluids due to the heterogeneous properties of PEG structures and interference from other substances present within the sample milieu. In this paper, we described a novel and simple approach to characterize PEGylated peptides in biological matrixes. The method takes advantage of the very different RP-HPLC binding properties that PEGylated peptides and proteins have relative to non-PEGylated peptides and proteins and other biological molecules. The method involves direct analysis of serum samples containing PEGylated products or treating them with acetonitrile to precipitate interfering serum proteins, while maintaining the conjugated molecule of interest in the supernatant for analysis. Combined with the established LC/ MS (19) approach for structural identification, our methodology demonstrated the precise fate of two model 20 kDa PEGylated peptides: PEGylated-PPA and PEGylated-glucagon, following incubation in biological fluids. The data clearly revealed a single C-terminal amino acid cleavage of the PPA backbone and a clip at C-terminal of Arg18 residue in glucagon. The spike and recovery evaluation has proved the method is valid in the 1 μg/mL sample concentration range. The availability of this methodology will greatly simplify the identification of PEGylated peptide/protein degradation mechanisms occurring in biological matrixes. On the basis of the robust analytical performance, the methodology may be generally applied to other PEG-conjugated therapeutic candidates, providing valuable high-resolution structural information that can be used to optimize molecular properties and support their pharmacological evaluation. ASSOCIATED CONTENT

S Supporting Information *

Additional informatioon as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. G

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