LC-MS/MS Identification of Impurities Present in Synthetic Peptide Drugs Dr Anna Meljon*, Dr Alan Thompson, Dr Osama Chahrour, and Dr John Malone Almac Sciences, 20 Seagoe Industrial Estate, Craigavon, Northern Ireland, BT63 5QD The presence of impurities, even at low concentrations, may affect the safety and efficacy of a drug substance (API). For most peptide pharmaceuticals, the API is defined and can be clearly characterised, however the identification of peptide related impurities can be challenging due to their complexity. A correct identification of the impurities is critical for the development and optimisation of the manufacturing process of the API material. Presented here is an example of a synthetic peptide API and related impurities characterisation performed at Almac. The peptide API and impurity sequences were elucidated using accurate mass measurements and MS/MS fragmentation. Using embedded algorithms in the MassHunter Software the accurate mass measurements allowed for the determination of the molecular formula of impurities. MS/MS fragmentation profiling allowed the identification of peptide and impurities sequences including the presence and the location of modifications. Accurate Mass Determination by LC-ESI-MS The analysis was performed using an Agilent 6530 Q-TOF tandem mass spectrometer coupled to an Agilent 1290 Infinity UHPLC system. The accurate mass was determined by MS analysis with an accuracy below 2 ppm. The peptide and impurities were observed as multiply charged ion species (Figure 1), which were recognised by the MassHunter software and deconvoluted to give the final parent masses (Figure 2). The software algorithm Extract by Molecular Feature was used to suggest modifications to the API which matched the impurity masses and isotopic distributions (Figure 3). Table 1: Example of Peptide Impurity Precursor Ion and Corresponding Product Ions. The Product Ions are Compared to the Theoretical Masses Resulting from Sequence Fragmentation Based on the accurate mass and the fragmentation pattern of the impurities, the software can suggest modifications of the API consistent with the impurity features and assign probability scores for each modification. Several impurities consistent with the loss of proline are displayed in Figure 3. Precursor Ion m/z  Z [M+H]+ 862.45  2 Figure 3: An Impurity Sequence Generated by BioConfirm X1LKVEX2SPX3X4SDX5INX6SPX7IEHX8P X1LKVEX2SPX3X4SDX5INX6SPX7IEHX X1LKVEX2SPX3X4SDX5INX6SX7IEHXP X1LKVEX2SX3X4SDX5INX6SPX7IEHXP Imp 1 Product Ion m/z (prod.) Diff (Bio, ppm) Sequence Z b8 8X9.5113 8X9.5091 2.5 KX1KVEX2SP 1 b11-NH3 11X2.6522 11X2.6477 3.8 KX1KVEX2SPX3X4S b18-NH3 6X8.3316 6X8.3338 -3.4 KX1KVEX2SPX3X4SDX5INX6SP 3 b20 10X3.5912 10X3.5944 -2.9 KX1KVEX2SPX3X4SDX5INX6SPX7I 2 b21 11X8.1181 11X8.1157 2.1 KX1KVEX2SPX3X4SDX5INX6SPX7IE b22 8X8.0948 8X8.0992 -5.3 KX1KVEX2SPX3X4SDX5INX6SPX7IEH y16 9X7.4416 9X7.4394 2.4 PX3X4SDX5INX6SPX7IEHX8 y19-H2O 104X.9891 104X.9874 1.6 EX2SPX3X4SDX5INX6SPX7IEHX8 y21-H2O 7X6.0456 7X6.0485 -3.8 KVEX2SPX3X4SDX5INX6SPX7IEHX8 y22 8X9.7471 8X9.7467 0.5 X1KVEX2SPX3X4SDX5INX6SPX7IEHX8 Figure 1: Mass Spectrum of a Peptide Impurity 5 x10 0.2 0.4 0.6 0.8 1 1.2 + Scan (6.819-6.869 min, 7 Scans) Imp1_MS_1.d 862.7812 1293.6664 335.0039 647.3374 1724.5485 Imp [M+3H]3+ Imp [M+2H]2+ Counts vs. Mass-to-Charge (m/z) 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 The resulting list of MS/MS fragments was processed using the ‘Extract by Molecular Feature’ algorithm of the MassHunter software and then matched with the theoretical list of daughter ions for the API within single digit ppm mass difference (Table 1). The accurate mass measurement determines the type of the modification whilst the MS/MS fragmentation defines the exact location of the modification. Peptide MS/MS daughter ions were compared with the list of theoretical product ions and the matched ions were annotated (Figure 4). The fragment ion abundance, the ion mass accuracy and the mass of an intact peptide are used to generate the hypothetical impurity sequence. The coverage of a peptide impurity sequence is displayed in Figure 5. Figure 2: Deconvoluted Mass Spectrum of a Peptide Impurity 5 x10 0.5 1 1.5 2 + Scan (6.819-6.869 min, 7 Scans) Imp1_MS_1.d Deconvoluted 2585.3210 2586.3239 2584.3169 2587.3261 2588.3281 2590.3309 2592.3144 Counts vs. Mass (amu) 2580 2581 2582 2583 2584 2585 2586 2587 2588 2589 2590 2591 2592 2593 2594 2595 Figure 5: Summary Sequence Coverage X1 K V E X2 S P X3 X4 D X5 I N X6 X7 H X8 b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 b16 b17 b18 b19 b20 b21 b22 y22 y21 y20 y19 y18 y17 y16 y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 Sequence Confirmed by LC-MS/MS Figure 4: Example of Interpreted Product Ion Spectrum with b and y Ion Assignments 3 x10 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 A(1-24) X1LKVEX2SPX3RSDYX4NASX5IEHX6 6.836: (M+3H)+3: + Product Ion (6.825, 6.834 ... min, 13 Scans) (862.45[z=3] -> **) Imp1_MSMS_2.d 862.44 2X1.10 y 2 1X9.10 b 1 9X1.98 b 17 2+ 10X7.05 b 19 2+ 4X0.14 y 3 7X2.05 y 21 3+ 11X2.57 y 21 2+ 5X3.23 y 4 6X7.86 b 12 2+ 994.97 1X3.15 324.23 12X1.63 y 11 1394.62 1493.69 1621.79 Counts vs. Mass-to-Charge (m/z) 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 Conclusion: Impurities present in peptide API can be determined using LC‐MS/MS with MassHunter software. The methodology utilised both accurate mass measurements and the fragmentation profiles of the analytes to provide the peptide impurity structure. Peptide Sequencing by LC-ESI-MS/MS The peptide and impurity precursor ions identified during the accurate mass determination were fragmented in the tandem mass spectrometer and the product ions analysed. The collision energy was tuned to obtain the most informative MS/MS spectrum containing b and y type ions. The fragment ions were analysed by ‘Find by Molecular Feature’ and ‘Define and Match Sequences’ algorithms to match the empirical fragment ions with the theoretical fragments. The ions were detected within a 20 ppm accuracy window. *Presenting author anna.meljon@almacgroup.com Note: X1-X8 amino acids were masked for IP purposes