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ZipChipTM Microfluidic CE-ESI Chip Separations Biotherapeutics
March 2016
// Actionable Intelligence – HPMS & On-board Analytics / Slide 1
March 2016
//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 // ZipChip CE-ESI MS: How Does it Work?
Injection
Cross
BGE
Background Electrolyte
(ESI-Compatible)
Waste
On-Line Desalting
Separation
Channel (10 cm)
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 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
// Actionable Intelligence – HPMS & On-board Analytics / Slide 3
March 2016
// ZipChip CE-ESI MS of Intact Infliximab Monoclonal Antibody
ZipChip HR
(22 cm separation)
Basic
Variants // Actionable Intelligence – HPMS & On-board Analytics / Slide 4
Acidic Variants Powerful MS information can be gained for
the CE-Separated Acidic and Basic Charge
Variants and associated Glycoforms
March 2016 // 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 //
Infliximab mAb - Deconvoluted MS for Charge Variants + Glycoforms
128 Da
127 Da
2-K
1-K
0-K
// Actionable Intelligence – HPMS & On-board Analytics / Slide 6
Two C-terminal lysines
Most highly-charged, fastest
CE migration time
Partial Truncation of
C-terminal lysines
Total Truncation of
C-terminal lysines
March 2016 // ZipChip CE-ESI MS for an IgG-2
2-K
0-K
130 Da
1 mg/mL IgG-2
10% 2-propanol 0.2%
acetic acid
1-K
131 Da
Very different Cterminal lysine
truncation charge
variant ratios relative
to Infliximab
0-K (major)
1-K
2-K
PTM
peak
// Actionable Intelligence – HPMS & On-board Analytics / Slide 7
March 2016 // ZipChip CE for Another IgG-2
0.5 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), 2220-2226.
// Actionable Intelligence – HPMS & On-board Analytics / Slide 8
March 2016 // 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 // 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 // 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), 2220-2226.
// Actionable Intelligence – HPMS & On-board Analytics / Slide 11
March 2016 // 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), 2220-2226.
// Actionable Intelligence – HPMS & On-board Analytics / Slide 12
March 2016 // DAR Distribution Comparison for ZipChip and iCE
Species CE-­‐MS Area % DAR-­‐0 7.7% DAR-­‐1 31.2% DAR-­‐2 45.9% DAR-­‐3 13.1% DAR-­‐4 2.1% Avg. DAR 1.7 Infusion Area % 8.6% 33.2% 39.0% 16.5% 2.7% 1.7 iCE Area % 5.8% 29.1% 44.8% 16.7% 3.6% 1.8 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), 2220-2226.
// Actionable Intelligence – HPMS & On-board Analytics / Slide 13
March 2016