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The world leader in serving scienceProprietary & Confidential
ZipChip™ and Thermo Scientific™ MS for CE/ESI-MS Analyses
Andrew Williamson
Applications specialist
2 Proprietary & Confidential
• The ZipChip system* uses integrated microfluidic technology to prepare, separate samples by capillary electrophoresis (CE), and then electrospray (ESI) analytes directly into a mass spectrometer (MS). Data collection, processing and reporting are through Xcalibur
• It is composed of the ZipChipinterface and the microfluidic chip
• The CE separation and ESI occur on the microfluidic chip
• ZipChip Interface directly mounts onto the front end of a mass spectrometer
• ZipChip system is compatible with a broad range of biomatrices such as growth media, cell lysates, blood, plasma, and urine
• The ZipChip system* uses integrated microfluidic technology to prepare, separate samples by capillary electrophoresis (CE), and then electrospray (ESI) analytes directly into a mass spectrometer (MS). Data collection, processing and reporting are through Xcalibur
• It is composed of the ZipChipinterface and the microfluidic chip
• The CE separation and ESI occur on the microfluidic chip
• ZipChip Interface directly mounts onto the front end of a mass spectrometer
• ZipChip system is compatible with a broad range of biomatrices such as growth media, cell lysates, blood, plasma, and urine
What is the ZipChip System?
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* ZipChip system is sold exclusively by Thermo Fisher in Europe and APAC
3 Proprietary & Confidential
Comprehensive Portfolio
ZipChip Interface ZipChip Autosampler ZipChip Assay KitsZipChips
• Two versions: Autosampler operation version and manual operation version
• Compatible with all Thermo ScientificTM
Exactive, Q Exactive Orbitrap MS, and LTQ Orbitrap MS instruments
• Disposable chips good for up to 125 injections per chip
• Two types: HR chip and HS chip
• 3 types of pre-made assay kits are designed for intact antibody, peptides, and metabolites analyses
• Fully automated and controlled by the ZipChip software
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4 Proprietary & Confidential
Simple ZipChip-MS Analysis Workflow
Place ZipChip and prepare the system
3
Simple Sample Prep
2
Set up sequence and collect CE-MS data
4
Select proper assay kit and ZipChip for your experiments
1
5 Proprietary & Confidential
Why the ZipChip System?
• The ONLY commercially available integrated and portable CE/ESI interface for MS
• Offers extremely rapid CE separations, nano-spray level sensitivity, and HRAM mass spectrometry in one platform
• Requires minimal sample preparation with on-chip desalting capability
• Consumes only picograms to nanograms of sample per analysis
Fast CE separation Nano Spray Sensitivity HRAM Mass Spectrometry
6 Proprietary & Confidential
Common Applications Performed on ZipChip-MS Platform
Metabolomics
Intact mAb/protein, and ADC characterization
mAb subunit analyses
Glycomics and glycoproteomics
Peptide mapping
7 Proprietary & Confidential
How ZipChip works
8 Proprietary & Confidential
• The ZipChip™ system uses integrated microfluidic technology to prepare, separate samples by capillary electrophoresis (CE), and then electrospray (ESI) analytes directly into a mass spectrometer (MS). It is composed of the ZipChip interface and the chip
• ZipChip Interface directly mounts onto the front end of a mass spectrometer
• The CE separation and ESI occur on the microfluidic chip
• ZipChip system is compatible with a broad range of biomatrices such as growth media, cell lysates, blood, plasma, and urine
• Each analysis only consumes a few nano liters of sample containing picograms to nano grams of analytes
• Only minimal sample preparation is needed
• The ZipChip™ system uses integrated microfluidic technology to prepare, separate samples by capillary electrophoresis (CE), and then electrospray (ESI) analytes directly into a mass spectrometer (MS). It is composed of the ZipChip interface and the chip
• ZipChip Interface directly mounts onto the front end of a mass spectrometer
• The CE separation and ESI occur on the microfluidic chip
• ZipChip system is compatible with a broad range of biomatrices such as growth media, cell lysates, blood, plasma, and urine
• Each analysis only consumes a few nano liters of sample containing picograms to nano grams of analytes
• Only minimal sample preparation is needed
ZipChip™ System Introduction
ZipChip HS ZipChip HRSeparation channel length (cm) 10 22Flowrate (nL/min) 150 150Maximum # of injections per chip 125 125On Chip De‐salting capability Yes YesIntegrated ESI Emitter Yes YesEEPROMS (recognize chip type and track usage) Yes YesRecommended use Small molecules or simple sample mixture Big molecules or complex sample mixtureTypical analysis time Up to 3 min Up to 15 min
9 Proprietary & Confidential
Anatomy of ZipChip
MS Inlet
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Glass microfluidic chip in PEEK housing
+HV1
+HV2
Well 1Waste
Well 2BGE
Well 3Sample
Well 4BGE
Well 2BGE
Well 3Sample
Well 1Waste
Well 4BGE
10 Proprietary & Confidential
+HV1
+HV2
Well 1Waste
Well 2BGE
Well 3Sample
Well 4BGE
Sample Injection
10 M rhodamine-6G in BGE + 100 mM ammonium acetate
11 Proprietary & Confidential
Sample Separation
• High voltage applied to Wells 2 and 4
• HV1 and HV2 determine field strength
• Field strength drives the ZipChip separation
+HV1
+HV2
Well 1Waste
Well 2BGE
Well 3Sample
Well 4BGE
6q‐ chargeη ‐ viscosity‐ hydrodynamicradius
1 2
For ZipChip analysis analytes must be positively charged in solution
Residual EOF++
++++++
+HV1
ESI Emitter
Well 1(Waste)
Well 3(Sample)
Well 2 (BGE)
‐‐
‐‐‐‐‐‐
(Electro-osmotic Flow)
Cations: Migrate to ESI emitter and are electrosprayed
Anions: Migrate to Well 2
Neutrals: Do not migrate. Pushed to waste by EOF
‐‐++
12 Proprietary & Confidential
Small Molecule Analysis/ Metabolomics
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ZipChip Separation of Amino Acids
2.5 M Promega Complete AA MixZipChip HSZipChip Metabolite KitExactive EMRField Strength: 1000 V/cm
1.8min
14 Proprietary & Confidential
ZipChip Analysis of Human Plasma
Chip type: ZipChip HSBGE: Metabolite kit (methanol/water/formic acid)Field Strength: 1000 V/cmInjection volume: 4 nL
• Simple sample prep• “dilute and shoot”
• Separation based only on charge and size
• Low nanomolar LODs
15 Proprietary & Confidential
Metabolite Analysis with ZipChipPeak # Migration Time
(minutes) m/z Assignment
1 0.54 89.1081 putrescine2 0.55 131.1296 agmatine3 0.57 120.9813 calcium adducts4 0.62 105.0036 salt adducts5 0.67 104.1075 choline6 0.74 147.1120 lysine7 0.75 133.0976 ornithine8 0.78 175.1184 arginine9 0.82 156.0760 histidine
10 0.92 76.0398 glycine11 0.99 90.0555 alanine12 1.11 118.0861 valine13 1.13 132.1025 isoleucine14 1.16 132.1025 leucine15 1.17 130.0865 pipecolic acid16 1.20 244.0927 cytidine17 1.26 120.0654 threonine18 1.31 116.0711 proline19 1.36 205.0962 tryptophan20 1.38 148.0608 glutamic acid21 1.43 268.1039 adenosine22 1.45 182.0807 tryosine23 1.49 134.0450 aspartic acid24 1.55 118.0862 betaine25 1.66 124.0399 picolinic acid26 1.94 238.0923 N-(1-Deoxy-1-fructosyl)glycine27 2.01 252.1082 N-(1-Deoxy-1-fructosyl)alanine28 2.06 294.1534 N-(1-Deoxy-1-fructosyl)isoleucine29 2.09 294.1534 N-(1-Deoxy-1-fructosyl)leucine30 2.12 280.1374 N-(1-Deoxy-1-fructosyl)valine31 2.35 268.1042 N-(1-Deoxy-1-fructosyl)serine32 2.37 328.1379 N-(1-Deoxy-1-fructosyl)phenylalanine33 2.64 295.1134 distichonic acid34 2.87 268.1038 Deoxyguanosine35 2.94 278.1243 N-(1-Deoxy-1-fructosyl)proline36 3.58 284.0972 guanosine37 4.03 137.0457 Hypoxanthine
10x dilution of fermentation media (beer)Metabolite Analysis KitZipChip HS
16 Proprietary & Confidential
Biopharmaceuticals
17 Proprietary & Confidential
The rapid separation and accurate identification of highly differently abundant charge variants can also be consistently achieved by the ZipChip™ system and Q Exactive™ platform
• Near baseline separation of intact NIST mAb charge variants with abundance ranging over 2 orders of magnitudes can be achieved by the ZipChip system
• High resolution accurate mass data of each Lysine variant is confidently obtained on Q Exactive™ Plus/HF/HF-X
• Glycoform with abundance as low as 0.16% of the base peak can be detected and identified
• All 5 major glycoforms from each of the three different Lysine variants are identified by BioPharma Finder
Intact NIST mAb Analysis in HMR Mode
4920 4940 4960 4980m/z
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Abu
ndan
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1004941.03
4946.44
4935.68
4951.80
4957.22
4945.27
4950.62
4939.84
4956.07
4961.46
4949.53
4944.114960.26
4965.69
NIST mAb + 0 Lys
NIST mAb + 1 Lys
NIST mAb + 2 Lys
+302.0 3.0Time (min)
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1004941.03
4781.695111.40
4632.30
4957.22
5293.894802.31
4945.27
5115.804785.75
5298.444636.194966.75 5138.04
4806.46
4949.535120.25
4789.87
5308.794640.21
4971.144810.97
5142.36
0.65nL of 0.1ug/ulNIST mAb injected
Protein Name Modification Average Mass(Da)
Theoretical Mass (Da)
Matched Mass Error (ppm)
Intensity Relative Abundance
RT (min)
NIST 1xG0F_G0F 148038.8 148037.1 11.5 1.28E+09 58.82NIST 1xG0F_G1F 148200.0 148199.3 5.0 2.18E+09 100.00NIST 1xG1F_G1F 148362.1 148361.2 6.0 1.82E+09 83.26NIST 1xG1F_G2F 148522.7 148523.5 5.8 1.00E+09 46.00NIST 1xG2F_G2F 148684.6 148685.7 7.2 4.84E+08 22.21NIST_plus1K 1xG0F_G0F 148165.6 148165.3 1.9 7.66E+07 3.51NIST_plus1K 1xG0F_G1F 148327.4 148327.4 0.1 1.39E+08 6.36NIST_plus1K 1xG1F_G1F 148489.7 148489.4 2.5 1.18E+08 5.40NIST_plus1K 1xG1F_G2F 148652.3 148651.7 3.6 5.97E+07 2.74NIST_plus1K 1xG2F_G2F 148811.9 148813.9 13.4 2.90E+07 1.33NIST_plus2K 1xG0F_G0F 148295.4 148293.5 13.1 1.04E+07 0.48NIST_plus2K 1xG0F_G1F 148456.9 148455.6 9.0 1.56E+07 0.72NIST_plus2K 1xG1F_G1F 148618.4 148617.5 5.6 1.51E+07 0.69NIST_plus2K 1xG1F_G2F 148779.4 148779.9 3.1 8.07E+06 0.37NIST_plus2K 1xG2F_G2F 148942.0 148942.0 0.4 3.49E+06 0.16
2.42
2.49
2.55
MS data was acquired on a QE HF with BioPharma OptionCE separation was achieved on ZipChip HR
18 Proprietary & Confidential
ZipChip Analysis of NIST Human Plasma, SRM 1950
0
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Concen
tration (m
icroMolar)
Highly Accurate Quantitation
ZipChip NIST Reference Values
* * * * *
* No NIST Reference Value
19 Proprietary & Confidential
0
50
100
1 2 3 4 5 6 7 8 9 10 11
The combination of ZipChip™ sample separation, Q Exactive™ Plus/HF/HF-X produced HRAM MS and MS/MS spectra, and BioPharma Finder™ software enables fast and accurate peptide identification
• Plug and play ZipChip™ delivers stable nano spray and nano spray level sensitivity
• CE-MS/MS analysis can be completed in 10 minutes
• Only a few nanograms of sample are sufficient for the analysis
• 98% sequence coverage based on MS/MS data for the light chain and heavy chain is confidently achieved
Proteins Number of MS Peaks
MS Peak Area
Sequence Coverage Abundance
NSIT mAb light chain 141 26.4% 100.0% 41.67%
NIST mAb heavy chain 339 60.5% 97.6% 56.35%
Unidentified 1441 12.6%
1.2 ng of sample injectedNIST mAb light chain
NIST mAb heavy chain
Unidentified
Peptide Mapping Analysis in Standard Mode
Rel
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Time (Min)
Electropherogram of Digested NIST mAb
MS data was acquired on a QE HF with BioPharma OptionCE separation was achieved on ZipChip HR
20 Proprietary & Confidential
Glycoproteomics
21 Proprietary & Confidential
Intact Analysis of Complex Glycoproteins
RT: 0.00 - 6.00
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time (min)
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100 5 mg/mL -1-acid glycoprotein, bovine serumZipChip Metabolite BGE, pH 2.2Exactive Plus EMR, Rs 17,500
Full Range, 500-4000 m/z
600-2000 m/z
Peptides and smaller proteins (~13.5 kDa)
-1-acid glycoprotein variants (30-34 kDa)
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z
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Rel
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2730.35
2754.632543.98
2706.08
2520.41
2778.902593.44
2496.75
2809.32
2834.94
1020.05776.22657.24 2978.471791.69
2862.73
964.33 2203.85 2340.36861.87 1123.36 1719.37 2054.601849.21 3030.001515.05
2408.15
1239.63 1427.95613.413092.65 3905.883738.403584.623392.203231.74
2520.91
2551.27 2750.081862.73
1781.251020.061940.87776.22 1735.59
2328.262018.08
1617.94798.84 2162.161500.48998.30 2047.221330.47816.25 1159.97 2783.041088.26
2705.902592.96
2224.66 2478.91 2848.27 3585.52945.76576.67 755.20 2505 2510 2515 2520 2525 2530 2535
m/z
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+12 Charge StateSpectrum obtained when all variants enter the mass spec at the same time
Spectrum of a single charge variant that has been separated before MS analysis
Severe spectral overlap of all variants combined
Clean spectrum from a single charge variant
22 Proprietary & Confidential
Glycoproteomics with ZipChip
• Glycopeptides naturally separate away from aglyco peptides
• In depth characterization of protein glycopeptides
• Improved separation resolution between glycopeptides compared to LC
• Reduced analysis times compared to LC
Alpha‐1‐acid glycoprotein tryptic digestZipChip HRThermo Q Exactive Plus
Khatri, K. et al; Microfluidic Capillary Electrophoresis-Mass Spectrometry for Analysis of Monosaccharides, Oligosaccharides, and Glycopeptides. Anal. Chem. 10.1021/acs.analchem.7b00875
23 Proprietary & Confidential
Top down and bottom up proteomics
24 Proprietary & Confidential
Combined Top-Down and Bottom-Up Analysis in One Platform
25 Proprietary & Confidential
Top-down + Bottom-up
67thP to H+4Phos
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time (min)
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Rel
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2.78
2.66
2.28 2.57
2.852.98
2.532.46
2.323.452.081.88
5Phos
4Phos
Acetyl
67thP to H
3Phos
RT: 0.00 - 8.00
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0Time (min)
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Rel
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eAb
unda
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2.58
3.69
3.83
2.67 4.552.74
2.804.94
3.20 4.083.48
Bottom Up Analysis of Cytochrome C Top Down Analysis of Beta Casein
Intact Protein Bottom-up Top-down
Proteoforms Sequence coverage Residue Cleavage Modifications
Cytochrome C 1 97% 50% 2
Carbonic anhydrase 1 97% 19% 1
KRAS 10 89% 15% 4
Beta-casein 11 86% 16% 7
• Fast runs, efficient separations, and rapid method switching enable multi-level characterization with no wasted time
26 Proprietary & Confidential
The ZipChip Advantage
• Efficient separations regardless of size
• Small molecules to large intact proteins
• Fast separations
• Sensitive and stable nano‐ESI
• Minimal sample prep
• No analyte labeling
• Separations based only on charge and size
• Fast and easy switching between analyte
classes
27 Proprietary & Confidential
Available Resources
Brochure
Spec sheets
Posters
TF.com page
Appl. Note
28 Proprietary & Confidential
The ZipChip™ system coupled with the Q Exactive™ platform can quickly analyze intact mAbs in native, partially denatured, and fully denatured conditions to support biotherapeutics characterizations under a diverse range of conditions
• CE/ESI-MS analysis can be completed within 3 minutes
• High resolution accurate mass spectra in intact native, partially denatured, and fully denatured states on the Q Exactive™ Plus/HF/HF-X with BioPharmaoption are confidently achieved
• Sample consumption can be as low as pico grams to nano grams
• Major glycoforms are identified by BioPharma Finder
G0/G0F
G0F/G0F
G0F/G1F
(G1F/G1F) or (G0F/G2F)
G1F/G2F1.0 ppm
3.9 ppm
1.0 ppm
7.5 ppm
2.4 ppm
0
20
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80
100 148056.78
148218.15
148379.38
147910.25 148542.25147800 148200 148600
Mass
Rel
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tens
ity
1 2 3Time (min)
0
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Rel
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0
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2.35
2.51
abc
Fully denatured
Partially denatured
z=26
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z=49
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020406080
100 5695.4
5923.25484.6
5288.7 6183.7
3022.6
3085.5
3151.2
3444.2
4936.2
4777.15106.4
4627.83897.25288.8
3797.4
Native
5660 5700 5740m/z
5695.4
5701.7
5707.75689.8 5714.0a
b
c
1 ng of sample injected
160 pg of sample injected
160 pg of sample injected
Full MS spectra
Electropherograms of intact Trastuzumab Deconvoluted spectrum
z=37
Intact mAb-- Trastuzumab Analysis in HMR mode
MS data was acquired on a QE HF-X with BioPharma OptionCE separation was achieved on ZipChip HR
29 Proprietary & Confidential
Electropherogram of intact infliximab:
0 2 4 6 8Time (min)
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2.6
4.53.5
0 Lys2 Lys1 Lys
5700 5720 5740m/z
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50100
Rel
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050
100 5722.8
5717.9
5713.0
zoom
z=26Full MS spectra
5000 6000m/z
050
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50
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Rel
ativ
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ance
050
100 5722.85510.8
5314.1 5951.7
5717.95506.2 5946.6
5309.5
5713.05941.45501.4
5305.0
z=26
z=25z=27
z=28 RT 2.6 min
RT 3.5 min
RT 4.5 min
2 Lys
0 Lys
1 Lys
0
20
40
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80
100148767.8
148511.9148929.2
148639.7
148673.3148801.2
148835.7 149092.6148964.9148564.0
149031.6 149199.3148311.6 149364.3 149481.5
148300 148500 148700 148900 149100 149300 149500Mass
0 2 4 6 8Time (min)0
20406080
100
Rel
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e Ab
unda
nce Sliding window deconvolution
with RespectTM algorithm
Rel
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tens
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[G0F/G0F] 2 Lys
[G0F/G1F] 2 Lys
[(G0F/G2F)/(G1F)2] 2 Lys
-Lys
-Lys
Deconvoluted MS spectrum
Theoretical and experimental masses of Infliximab
glycoforms of Lysine variants
Lys-Variant Glycoform Theor. average mass Exp. average mass Mass (ppm)2 Lys G0F/G1F 148930.7 148929.2 9.82 Lys G0F/G0F 148768.5 148767.8 4.82 Lys (G1F)2 or (G0F/G2F) 149092.8 149092.6 1.31 Lys G0F/G0F 148640.3 148639.7 4.31 Lys G0F/G1F 148802.5 148801.2 8.61 Lys (G1F)2 or (G0F/G2F) 148964.6 148964.9 -1.80 Lys G0F/G0F 148512.2 148511.9 1.60 Lys G0F/G1F 148674.3 148673.3 6.80 Lys (G1F)2 or (G0F/G2F) 148836.5 148835.7 5.1
The ZipChip™ system coupled with the Q Exactive™ platform is unique and powerful to separate and identify different intact antibody charge variants in native, partially denatured, and denatured conditions
• Baseline separation of intact mAb charge variants resulting from different levels of Lys-clipping can be achieved within three minutes by ZipChip™ system
• High resolution accurate mass spectra of all lysine variants are confidently detected on Q Exactive Plus/HF/HF-X with BioPharma option
• Three major glycoforms from each of the three lysine variants are identified by BioPharma Finder with mass accuracies better than 10 ppm
3 ng of sample injected
Intact mAb– Infliximab Analysis in HMR mode
MS data was acquired on a QE HF-X with BioPharma OptionCE separation was achieved on ZipChip HR
30 Proprietary & Confidential
Fast, sensitive, and accurate antibody subunit analysis can be accomplished by the ZipChip™ system and Thermo Scientific™ Q Exactive™ Plus or HF/HF-X platform
• Separation of infliximab mAb subunits can be achieved in 3 minutes by ZipChip™ system
• The sliding window method combined with Xtract™ deconvolution algorithm in BioPharma Finder ™ software enables monoisotopic mass determination of each subunit
• Lysine variants and their major glycoforms of the subunits can be identified by BioPharma Finder software
IdeS digested + reduced infliximabIdeS digested infliximab
Subunit Monoisotopic theo. mass Experimental mass Mass (ppm)Light chain 23424.3946 23424.4671 3.1
Fd' 25631.5325 25631.5644 1.2Fc/2 G0F 25316.5863 25316.6345 1.9Fc/2 G1F 25478.6391 25478.5753 2.5
(Fc/2 G0F) -Lys 25188.49131 25188.5763 3.3(Fc/2 G1F) -Lys 25350.54413 25350.5246 0.8
F(ab')2 98146.3 (av.) 98146.8 -4.8Fc/2 G0F (2 SS bonds) 25312.5550 25312.5524 0.1Fc/2 G1F (2 SS bonds) 25474.6078 25474.5138 3.7
(Fc/2 G0F) -Lys (2 SS bonds) 25184.4600 25184.4311 1.1(Fc/2 G1F)- Lys (2 SS bonds) 25346.5128 25346.4730 1.6
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2.11
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2.26
(Fc/2) -LysF(ab’)2
Fc/2
120 k 15 k
z=44
z=45z=42
z=43
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2283.52231.7
2182.0z=41
z=46
z=48
Deconvolutionwith Respect 98146.8
98000 100000Mass
0
25312.552425184.4311
25474.513825346.4730
25100 25300 25500Mass
20406080
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Rel
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(Fc/2 G1F) -LysFc/2 G1F
F(ab’)2
Deconvolutionwith Xtract
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Rel
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3.42 3.48
3.06
LC
Fd’(Fc/2) -LysFc/2
120 k
800 900 1000 1100m/z0
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845.43905.71
975.301014.31
1102.43
901.17970.37
1009.231096.90
841.13 (Fc/2) -Lys
Fc/2
LC
Fd’
1000 1500m/z0
50
1000
50
100
Rel
ativ
e A
bund
ance
885.41 1026.871069.61
1166.81
938.54
1066.351172.93
1303.14
869.10
23424.4671 25631.5644
20406080
100
Rel
ativ
e In
tens
ity
Mass 2500024000
Fd’LC
(Fc/2 G0F) -Lys
Fc/2 G0F(Fc/2 G1F) -Lys
Fc/2 G1F
0
1200 1400m/z0
50
1000
50
100
Rel
ativ
e A
bund
ance
1327.21401.1
1261.01483.3
1201.0
(Fc/2) -Lys
1334.11267.4
1408.21207.01490.9
Fc/2
IdeS
dige
st
IdeS
dige
st
plus
re
duct
ion
mAb Subunits Analysis in Protein mode
MS data was acquired on a QE HF-X with BioPharma OptionCE separation was achieved on ZipChip HR
31 Proprietary & Confidential
0
50
100 150931.7151091.8152048.7
151888.7151252.7149974.5150134.3
153007.0152210.5
150294.9152845.5
153168.3149176.3149015.4151314.3
153964.5149336.8154126.3150358.5 152272.1151411.8 152371.5 153802.9150453.7
154921.9148220.6150580.1 153328.4 155143.3151538.8149499.1 154150.3149559.6148379.0 154758.8 155308.0
148871.3154185.4152497.4
154282.5
148440.6 151744.1 155805.4153467.9 154661.2
155718.9
155474.7
154922.8
148500 150000 151500 153000 154500 156000
2.0 3.0 4.0 5.00
50
1003.34
1.0
Heterogenetic ADCs can be successfully characterized within 1 minute without sample pre-treatment by ZipChip™system and Q Exactive™ Plus/HF/HF-X, and the powerful BioPharma Finder™ software
• Powerful sliding window capability enabled by BioPharma Finder
• All of the different forms of Trastuzumab Emtansine can be analyzed in less than 40 seconds with only 3 nanograms of sample injected
• No sample pre-treatment required
• The calculated average DAR values are consistent with previous published data
Antibody-Drug Conjugate (ADC) Analysis in HMR Mode
4600 4650 4700 4750 4800 4850 4900 4950 5000 5050 5100 5150 5200 5250 5300 5350 5400 5450 55000
50
100 5037.424874.895005.444905.77
4844.05 5069.29
5042.76
4911.014967.86
5074.72 5178.01 5205.514722.68 5101.284752.56 4813.01 5106.594782.455244.08
5145.035249.65
4692.75 5133.15 5271.445277.07
4995.22
5362.97 5391.495282.564608.56 5397.234637.63 5328.825304.574657.80 5431.315436.84
5471.365499.94
Electropherogram of Trastuzumab Emtansine
MS Full Spectrum
Deconvoluted Spectrum
M/Z
Rel
ativ
e A
bund
ance
DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 DAR 6 DAR 7 DAR 8
G0F/G1F DAR Form Mass Accurancy (ppm) Relative abundanceDAR0 10.72 9.95DAR1 7.45 33.47DAR2 1.23 58.97DAR3 2.37 79.90DAR4 5.63 69.92DAR5 9.08 49.14DAR6 13.89 28.97DAR7 9.93 12.64DAR8 8.41 2.83
Average Drug‐to‐Antibody Ratio (DAR) is 3.47
MS data was acquired on a QE HF with BioPharma OptionCE separation was achieved on ZipChip HR
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