Monitoring Protein PEGylation with Ion Exchange Chromatography

Transcription

1 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

2 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

3 Potential Challenges Positional Isomerism Site Specification Separation of Complex Mixtures 3

4 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

5 Reagents Branched Amine-Reactive PEG Lysozyme six potential lysine sites, 14.3 kd RNase A ten potential lysine sites, 13.7 kd 5

6 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

7 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

8 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

9 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

10 Literature Separation of PEGylated Lysozyme Biotechnol. J. 21, 5,

11 PEGylated Lysozyme Separation from a Vendor s Application Note 11

12 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

13 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 Minutes 26 13

14 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 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 % signal offset Minutes 1:5 PEG/Lysozyme 1:1 PEG/Lysozyme Lysozyme 14

15 PEGylated Lysozyme Deconvoluted MS Spectra Lysozyme :1 PEG:Lysozyme 1:5 PEG:Lysozyme :3 PEG:Lysozyme 1:1 PEG:Lysozyme m/z 3:1 PEG:Lysozyme 5:1 PEG:Lysozyme 1:1 PEG:Lysozyme 15

16 Expected Masses Correlate with Observed Masses Protein Expected Mass (kda) Observed Mass (kda) Lysozyme Lysozyme + 1 PEG Lysozyme + 2 PEG Lysozyme + 3 PEG Lysozyme + 4 PEG Lysozyme + 5 PEG Lysozyme + 6 PEG 28.1 Not Detected 16

17 Degree of PEGylation Tracked by Cation Exchange Protein Peak Height (mau) 17

18 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 Minutes

19 PEGylated RNase A Deconvoluted MS Spectra 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: 1.54E :1 PEG:RNase A NL: 1.65E :1 PEG:RNase A m/z 19

20 Expected Masses Correlate with Observed Masses Protein Expected Mass (kda) Observed Mass (kda) Ribonuclease A , 13.7 Ribonuclease A + 1 PEG Ribonuclease A + 2 PEG Ribonuclease A + 3 PEG Ribonuclease A + 4 PEG Ribonuclease A + 5 PEG Ribonuclease A + 6 PEG Ribonuclease A + 7 PEG 29.8 Not Detected 2

21 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

22 22 Thank you for your attention!