Monitoring Protein PEGylation with Ion Exchange Chromatography Peter Yu, Deanna Hurum, Leo Wang, Terry Zhang, and Jeffrey Rohrer 245 th ACS National Meeting & Exposition April 9, 213 OT7618_E 4/13S 1 The world leader in serving science
PEGylation of Therapeutic Proteins Polyethylene glycol (PEG)ylation is used to modify therapeutic proteins. Improved stability Reduced circulatory clearance Potential for smaller doses/greater time between doses It is used in numerous pharmaceutical agents for delivery. Most often, N-Hydroxysuccinimide (NHS) is used to react with lysine amines to form a covalent attachment with the protein. 2
Potential Challenges Positional Isomerism Site Specification Separation of Complex Mixtures 3
Ion Exchange and PEGylated proteins Two model proteins: Lysozyme RNase A A small but commercially available PEG was used to modify these model proteins. The reaction conditions, varying both time and the reactant ratio, were investigated. 4
Reagents Branched Amine-Reactive PEG Lysozyme six potential lysine sites, 14.3 kd RNase A ten potential lysine sites, 13.7 kd 5
Sample Preparation Dissolve 1 mg protein in 1 ml of phosphate-buffered saline. Dispense appropriate volume of PEGylation reagent into protein solution to achieve the desired reactant molar ratios. Incubate at room temperature for 3 min. Desalt using.5 ml Thermo Scientific Zeba Spin Desalting Columns to remove buffers and unreacted PEGylation reagent. 6
Instrumentation Thermo Scientific Dionex UltiMate 3 BioRS System consisting of: SRD-36 Integrated Solvent and Degasser Rack DGP-36RS Biocompatible Dual-Gradient Rapid Separation Pump TCC-3SD Thermostatted Column Compartment WPS-3TBRS Thermostatted Biocompatible Rapid Separation Autosampler VWD-34 Variable Wavelength Detector 7
Chromatographic Method Column: Thermo Scientific ProPac SCX-1G Guard (2 5 mm), ProPac SCX-1 Analytical (2 25 mm) Mobile Phases: A) 2 mm MES, ph 6.1 B) 2 mm MES, 1M NaCl, ph 6.1 Gradient: Flow Rate: Inj. Volume: 1 µl Detection: 3% B from to 3 min, equilibration at % B for 1 min before injection.25 ml/min UV, 28 nm 8
Mass Spectrometry The PEGylation reaction mixture was desalted and injected on a Thermo Scientific BioBasic 8 HPLC Column (5 µm, 1. mm). MS confirmation was performed on a Thermo Scientific Q Exactive Orbitrap mass spectrometer with 14, mass resolution. The Q Exactive was operated in positive full-scan mode with scan range from to 3 m/z. Mass spectra deconvolution was accomplished using the Xtract Algorithm of the Thermo Scientific Xcalibur software. 9
Literature Separation of PEGylated Lysozyme Biotechnol. J. 21, 5, 477 483. 1
PEGylated Lysozyme Separation from a Vendor s Application Note 11
ProPac IEX Resin with Grafted Polymeric Functionalized Chains 1 µm Nonporous Polymeric Beads Polymeric Grafts Boundary (Cross-Linked Hydrophilic Layer) Core (Highly Cross-Linked EVB-DVB) EVB-DVB: Ethylvinylbenzene-Divinylbenzene 12
Changes in Lysozyme PEGylation with Reaction Time UV_VIS_1 WVL:28 nm 18 Columns: ProPac SCX-1G Guard, 2 5 mm ProPac SCX-1 Analytical, 2 25 mm Mobile Phases: A) 2 mm MES, ph 6.1 B) 2 mm MES, 1M NaCl, ph 6.1 Gradient: Flow Rate: Inj. Volume: 1 µl Samples: 3% B from to 3 min Equilibration at % B for 1 min before injection.25 ml/min Lysozyme (black) Lysozyme reacted with PEG for 5 min (blue) Lysozyme reacted PEG for 3 min (red) mau -2 4.3 7.5 12.5 17.5 22.5 Minutes 26 13
Changes in PEGylation with Reactant Ratios Lysozyme Columns: ProPac SCX-1G Guard, 2 5 mm ProPac SCX-1 Analytical, 2 25 mm Mobile Phases: A) 2 mm MES, ph 6.1 B) 2 mm MES, 1M NaCl, ph 6.1 54 5 4 Gradient: Flow Rate: 3% B from to 3 min Equilibration at % B for 1 min before injection.25 ml/min 3 Inj. Volume: 1 µl 2 1 Lysozyme 1:1 PEG/Lysozyme 5:1 PEG/Lysozyme mau 3:1 PEG/Lysozyme 1:1 PEG/Lysozyme 1:3 PEG/Lysozyme -1.. 8. 16. 24. 26. 1% signal offset Minutes 1:5 PEG/Lysozyme 1:1 PEG/Lysozyme Lysozyme 14
PEGylated Lysozyme Deconvoluted MS Spectra 1433.88 1433.84 Lysozyme 1433.83 1433.83 1661.17 1661.18 1661.18 18915.52 1:1 PEG:Lysozyme 1:5 PEG:Lysozyme 1433.85 1661.18 18915.54 18915.53 18915.53 21221.88 21221.87 1:3 PEG:Lysozyme 1:1 PEG:Lysozyme 1661.17 23527.24 21221.87 23527.23 18915.52 25789.56 23526.21 21221.87 18915.53 25789.56 15 2 25 3 m/z 3:1 PEG:Lysozyme 5:1 PEG:Lysozyme 1:1 PEG:Lysozyme 15
Expected Masses Correlate with Observed Masses Protein Expected Mass (kda) Observed Mass (kda) Lysozyme 14.3 14.3 Lysozyme + 1 PEG 16.6 16.6 Lysozyme + 2 PEG 18.9 18.9 Lysozyme + 3 PEG 21.2 21.2 Lysozyme + 4 PEG 23.5 23.5 Lysozyme + 5 PEG 25.8 25.8 Lysozyme + 6 PEG 28.1 Not Detected 16
Degree of PEGylation Tracked by Cation Exchange Protein Peak Height (mau) 17
Changes in PEGylation with Reactant Ratios RNase A Columns: ProPac SCX-1G Guard, 2 5 mm, ProPac SCX-1 Analytical, 2 25 mm Mobile Phases: A) 2 mm MES, ph 6.1 B) 2 mm MES, 1M NaCl, ph 6.1 Gradient: 3% B from to 3 min Equilibration at % B for 1 min before injection Flow Rate:.25 ml/min 6 5,6 4 Inj. Volume: 1 µl 2, 3 1 RNase A 1:1 PEG/RNase A 5:1 PEG/RNase A mau 3:1 PEG/RNase A 1:1 PEG/RNase A 1:3 PEG/RNase A 1:5 PEG/RNase A 1:1 PEG/RNase A RNase A 1% signal offset -1.8 3. 1 Minutes 2 26 18
PEGylated RNase A Deconvoluted MS Spectra 11533.26 11532.27 13424.21 13682.37 1394.35 13681.26 13682.27 13681.27 11533.26 13681.27 11533.26 15986.63 18292.98 18292.97 2598.33 18292.97 13838.6 2598.34 16144.89 13681.28 2294.7 2598.33 2293.7 18292.98 16143.88 13839.65 25211.5 2293.67 2521.3 NL: 5.31E4 RNase A NL: 6.15E6 1:1 PEG:RNase A NL: 3.62E6 1:5 PEG:RNase A NL: 2.99E6 1:3 PEG:RNase A NL: 1.14E6 1:1 PEG:RNase A NL: 2.87E5 3:1 PEG:RNase A NL: 1.54E5 27926.92 5:1 PEG:RNase A NL: 1.65E5 2598.32 27515.4 1:1 PEG:RNase A 15 2 25 3 m/z 19
Expected Masses Correlate with Observed Masses Protein Expected Mass (kda) Observed Mass (kda) Ribonuclease A 13.7 13.4, 13.7 Ribonuclease A + 1 PEG 16. 16. Ribonuclease A + 2 PEG 18.3 18.3 Ribonuclease A + 3 PEG 2.6 2.6 Ribonuclease A + 4 PEG 22.9 22.9 Ribonuclease A + 5 PEG 25.2 25.2 Ribonuclease A + 6 PEG 27.5 27.5 Ribonuclease A + 7 PEG 29.8 Not Detected 2
Conclusions PEGylated proteins were separated using SCX chromatography, revealing numerous isomers. These isomers were separated by the degree of PEGylation as confirmed by MS. PEGylation can be monitored as a function of reaction conditions, allowing reaction optimization for the desired product. SCX chromatography is shown to be a powerful method for characterizing modified protein mixtures with multiple positional isomers. 21
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