ZipChipTM. Microfluidic CE-ESI Chip Separations Biotherapeutics. March // Actionable Intelligence HPMS & On-board Analytics / Slide 1

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1 ZipChipTM Microfluidic CE-ESI Chip Separations Biotherapeutics March 2016 // Actionable Intelligence HPMS & On-board Analytics / Slide 1 March 2016

2 //ZipChip Biotherapeutics Analysis Summary Fast, high-resolution CE in runs of three minutes or less for large IgGs and ADCs Many charge variant/ptm species separated and identified. Full power of MS utilized Significantly improved CE separations and analysis times compared to HPLC For ADCs, accurate DAR distributions are achieved with MS detection Minimal sample preparation required due to ZipChip on-line desalting Low volume nl injections advantageous for precious samples Negligible carryover in ZipChip CE Optimized CE method available on the ZipChip HR for intact biologics analysis. Potential for top-down proteomics in the same experiment Can perform complimentary Peptide Mapping experiment using the ZipChip HS (10 cm separation) and/or the ZipChip HR (22 cm separation) // Actionable Intelligence HPMS & On-board Analytics / Slide 2 March 2016

3 // ZipChip CE-ESI MS: How Does it Work? Injection Cross BGE Background Electrolyte (ESI-Compatible) Waste Separation Channel (10 cm) On-Line Desalting Highly uniform and stable surface coating efficient separations with reduced electroosmotic flow ESI MS Inlet: 0 kv ZipChip developed by the J. Michael Ramsey Group at University of North Carolina over the last 10 years // Actionable Intelligence HPMS & On-board Analytics / Slide 3 CE separation achieved through differences in electrophoretic mobility which is governed by charge and size of analyte Batz, Mellors, Alarie, Ramsey, Anal Chem, 2014, 86, 3493 March 2016

4 // ZipChip CE-ESI MS of Intact Infliximab Monoclonal Antibody ZipChip HR (22 cm separation) Basic Variants Acidic Variants Powerful MS information can be gained for the CE-Separated Acidic and Basic Charge Variants and associated Glycoforms // Actionable Intelligence HPMS & On-board Analytics / Slide 4 March 2016

5 // ZipChip CE-ESI MS of Intact Infliximab Monoclonal Antibody ZipChip HR (22 cm separation) Glycoforms of each Charge Variant can be identified from the deconvoluted mass spectrum // Actionable Intelligence HPMS & On-board Analytics / Slide 5 March 2016

6 // Infliximab mab - Deconvoluted MS for Charge Variants + Glycoforms 128 Da 2-K Two C-terminal lysines Most highly-charged, fastest CE migration time 127 Da 1-K Partial Truncation of C-terminal lysines 0-K Total Truncation of C-terminal lysines // Actionable Intelligence HPMS & On-board Analytics / Slide 6 March 2016

7 // ZipChip CE-ESI MS for an IgG-2 0-K 1 mg/ml IgG-2 10% 2-propanol 0.2% acetic acid 130 Da 2-K 131 Da 1-K Very different C- terminal lysine truncation charge variant ratios relative to Infliximab 1-K 2-K PTM peak 0-K (major) // Actionable Intelligence HPMS & On-board Analytics / Slide 7 March 2016

8 // ZipChip CE for Another IgG mg/ml IgG-2 10% 2-propanol 0.2% Acetic Acid Deconvolution of the above spectrum Various PTMs and associated glycoforms separated by ZipChip CE and identified - MS mining would reveal more important IgG characterization information Fast CE analysis just over 2 min Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 8 March 2016

9 // ZipChip CE-ESI MS of IgG Tryptic Digest Peptide Mapping IgG2 Monoclonal Antibody Tryptic Digest, 0.1 mg.ml BGE: 50% methanol, 2% formic acid (ph2.2) No Additional Sample Preparation ZipChip HS (10 cm separation) Increased coverage of light and heavy peptides relative to LC/MS Peptide Mapping CE analysis time less than 3 minutes Can extend run time using ZipChip HR for potentially more peptide IDs Peptide Mapping data highly complementary to the Intact data 9 // Actionable Intelligence HPMS & On-board Analytics / Slide 9 March 2016

10 // ZipChip CE-ESI MS of Intact Antibody Drug Conjugate Antibody Drug Conjugate, 1 mg/ml ZipChip HR (22 cm separation channel) BGE: 10% IPA, 0.2% acetic acid (ph 3.5) Excellent CE separations observed for ADCs in fast analysis time Very challenging experiment for LC/MS Redman, Mellor, Starkey, Ramsey, Anal. Chem., 2016, 88(4), 2220 // Actionable Intelligence HPMS & On-board Analytics / Slide 10 March 2016

11 // ZipChip CE-ESI MS of an Intact Antibody Drug Conjugate 1 mg/ml ADC 10% 2-propanol 0.2% Acetic Acid A wealth of ADC conjugates, glycoforms and PTMs revealed by ZipChip CE-ESI MS CE analysis time less than 3 min. Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 11 March 2016

12 // Deconvolution of DAR Spectra Overlaid spectra from each DAR peak in the separation Avg. mass shift due to conjugation: 3145 Da Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 12 March 2016

13 // DAR Distribution Comparison for ZipChip and ice Species CE- MS Area % Infusion Area % ice Area % DAR % 8.6% 5.8% DAR % 33.2% 29.1% DAR % 39.0% 44.8% DAR % 16.5% 16.7% DAR % 2.7% 3.6% Avg. DAR Close agreement between ZipChip CE-ESI MS and established ice DAR distributions ice data was provided by collaborators Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 13 March 2016