ZipChip and Thermo Scientific MS for CE/ESI-MS Analyses Andrew - - PowerPoint PPT Presentation

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ZipChip and Thermo Scientific MS for CE/ESI-MS Analyses Andrew - - PowerPoint PPT Presentation

Feb 07, 2023 314 likes •647 views

ZipChip and Thermo Scientific MS for CE/ESI-MS Analyses Andrew Williamson Applications specialist The world leader in serving science Proprietary & Confidential What is the ZipChip System? The ZipChip system* uses integrated

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The world leader in serving science

Proprietary & Confidential

ZipChip™ and Thermo Scientific™ MS for CE/ESI-MS Analyses

Andrew Williamson Applications specialist

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  • 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 ZipChip

interface and the microfluidic chip

  • The CE separation and ESI occur on

the microfluidic chip

  • ZipChip Interface directly mounts
  • nto 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 ZipChip

interface and the microfluidic chip

  • The CE separation and ESI occur on

the microfluidic chip

  • ZipChip Interface directly mounts
  • nto 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

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Comprehensive Portfolio

ZipChip Interface ZipChip Autosampler ZipChip Assay Kits ZipChips

  • Two versions:

Autosampler operation version and manual

  • peration 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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Simple ZipChip-MS Analysis Workflow

Place ZipChip and prepare the system

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Simple Sample Prep

2

Set up sequence and collect CE-MS data

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Select proper assay kit and ZipChip for your experiments

1

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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

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Common Applications Performed on ZipChip-MS Platform

Metabolomics Intact mAb/protein, and ADC characterization mAb subunit analyses Glycomics and glycoproteomics Peptide mapping

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How ZipChip works

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  • 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 pico grams 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 pico grams to nano grams of analytes

  • Only minimal sample preparation is

needed

ZipChip™ System Introduction

ZipChip HS ZipChip HR

Separation channel length (cm)

10 22

Flowrate (nL/min)

150 150

Maximum # of injections per chip

125 125

On Chip De‐salting capability

Yes Yes

Integrated ESI Emitter

Yes Yes

EEPROMS (recognize chip type and track usage)

Yes Yes

Recommended use

Small molecules or simple sample mixture Big molecules or complex sample mixture

Typical analysis time

Up to 3 min Up to 15 min

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Anatomy of ZipChip

MS Inlet

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Glass microfluidic chip in PEEK housing +HV1 +HV2

Well 1 Waste Well 2 BGE Well 3 Sample Well 4 BGE Well 2 BGE Well 3 Sample Well 1 Waste Well 4 BGE

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+HV1 +HV2 Well 1 Waste Well 2 BGE Well 3 Sample Well 4 BGE

Sample Injection

10 M rhodamine-6G in BGE + 100 mM ammonium acetate

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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 1 Waste Well 2 BGE Well 3 Sample Well 4 BGE

  • 6

q ‐ charge η ‐ viscosity ‐ hydrodynamic radius 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

‐ +

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Small Molecule Analysis/ Metabolomics

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ZipChip Separation of Amino Acids

2.5 M Promega Complete AA Mix

ZipChip HS ZipChip Metabolite Kit Exactive EMR Field Strength: 1000 V/cm

1.8 min

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ZipChip Analysis of Human Plasma

Chip type: ZipChip HS BGE: Metabolite kit (methanol/water/formic acid) Field Strength: 1000 V/cm Injection volume: 4 nL

  • Simple sample prep
  • “dilute and shoot”
  • Separation based only
  • n charge and size
  • Low nanomolar LODs
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Metabolite Analysis with ZipChip

Peak # Migration Time (minutes) m/z Assignment 1 0.54 89.1081 putrescine 2 0.55 131.1296 agmatine 3 0.57 120.9813 calcium adducts 4 0.62 105.0036 salt adducts 5 0.67 104.1075 choline 6 0.74 147.1120 lysine 7 0.75 133.0976

  • rnithine

8 0.78 175.1184 arginine 9 0.82 156.0760 histidine 10 0.92 76.0398 glycine 11 0.99 90.0555 alanine 12 1.11 118.0861 valine 13 1.13 132.1025 isoleucine 14 1.16 132.1025 leucine 15 1.17 130.0865 pipecolic acid 16 1.20 244.0927 cytidine 17 1.26 120.0654 threonine 18 1.31 116.0711 proline 19 1.36 205.0962 tryptophan 20 1.38 148.0608 glutamic acid 21 1.43 268.1039 adenosine 22 1.45 182.0807 tryosine 23 1.49 134.0450 aspartic acid 24 1.55 118.0862 betaine 25 1.66 124.0399 picolinic acid 26 1.94 238.0923 N-(1-Deoxy-1-fructosyl)glycine 27 2.01 252.1082 N-(1-Deoxy-1-fructosyl)alanine 28 2.06 294.1534 N-(1-Deoxy-1-fructosyl)isoleucine 29 2.09 294.1534 N-(1-Deoxy-1-fructosyl)leucine 30 2.12 280.1374 N-(1-Deoxy-1-fructosyl)valine 31 2.35 268.1042 N-(1-Deoxy-1-fructosyl)serine 32 2.37 328.1379 N-(1-Deoxy-1-fructosyl)phenylalanine 33 2.64 295.1134 distichonic acid 34 2.87 268.1038 Deoxyguanosine 35 2.94 278.1243 N-(1-Deoxy-1-fructosyl)proline 36 3.58 284.0972 guanosine 37 4.03 137.0457 Hypoxanthine

10x dilution of fermentation media (beer) Metabolite Analysis Kit ZipChip HS

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Biopharmaceuticals

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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

  • btained 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 4980 m/z 20 40 60 80 100 20 40 60 80 100 R elative A bundance 20 40 60 80 100 4941.03 4946.44 4935.68 4951.80 4957.22 4945.27 4950.62 4939.84 4956.07 4961.46 4949.53 4944.11 4960.26 4965.69

NIST mAb + 0 Lys NIST mAb + 1 Lys NIST mAb + 2 Lys

+30

2.0 3.0 Time (min) 20 40 60 80 100 Relative Abundance 2.55 2.52 2.49

0 Lys 1 Lys 2 Lys 4600 4800 5000 5200 5400 m/z 50 100 50 100 R elative A bundance 50 100 4941.03 4781.69 5111.40 4632.30 4957.22 5293.89 4802.31 4945.27 5115.80 4785.75 5298.44 4636.19 4966.75 5138.04 4806.46 4949.53 5120.25 4789.87 5308.79 4640.21 4971.14 4810.97 5142.36 0.65nL of 0.1ug/ul NIST 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 58.82 NIST 1xG0F_G1F 148200.0 148199.3 5.0 2.18E+09 100.00 NIST 1xG1F_G1F 148362.1 148361.2 6.0 1.82E+09 83.26 NIST 1xG1F_G2F 148522.7 148523.5 5.8 1.00E+09 46.00 NIST 1xG2F_G2F 148684.6 148685.7 7.2 4.84E+08 22.21 NIST_plus1K 1xG0F_G0F 148165.6 148165.3 1.9 7.66E+07 3.51 NIST_plus1K 1xG0F_G1F 148327.4 148327.4 0.1 1.39E+08 6.36 NIST_plus1K 1xG1F_G1F 148489.7 148489.4 2.5 1.18E+08 5.40 NIST_plus1K 1xG1F_G2F 148652.3 148651.7 3.6 5.97E+07 2.74 NIST_plus1K 1xG2F_G2F 148811.9 148813.9 13.4 2.90E+07 1.33 NIST_plus2K 1xG0F_G0F 148295.4 148293.5 13.1 1.04E+07 0.48 NIST_plus2K 1xG0F_G1F 148456.9 148455.6 9.0 1.56E+07 0.72 NIST_plus2K 1xG1F_G1F 148618.4 148617.5 5.6 1.51E+07 0.69 NIST_plus2K 1xG1F_G2F 148779.4 148779.9 3.1 8.07E+06 0.37 NIST_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 Option CE separation was achieved on ZipChip HR

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ZipChip Analysis of NIST Human Plasma, SRM 1950

50 100 150 200 250 300 350

Concentration (microMolar)

Highly Accurate Quantitation

ZipChip NIST Reference Values

* * * * *

* No NIST Reference Value

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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 injected

NIST mAb light chain NIST mAb heavy chain Unidentified

Peptide Mapping Analysis in Standard Mode

Relative Abundance Time (Min) Electropherogram of Digested NIST mAb MS data was acquired on a QE HF with BioPharma Option CE separation was achieved on ZipChip HR

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Glycoproteomics

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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 6.0 Time (min) 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Relative Abundance 10 20 30 40 50 60 70 80 90 100

5 mg/mL -1-acid glycoprotein, bovine serum ZipChip Metabolite BGE, pH 2.2 Exactive 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 4000 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 2730.35 2754.63 2543.98 2706.08 2520.41 2778.90 2593.44 2496.75 2809.32 2834.94 1020.05 776.22 657.24 2978.47 1791.69 2862.73 964.33 2203.85 2340.36 861.87 1123.36 1719.37 2054.60 1849.21 3030.00 1515.05 2408.15 1239.63 1427.95 613.41 3092.65 3905.88 3738.40 3584.62 3392.20 3231.74 2520.91 2551.27 2750.08 1862.73 1781.25 1020.06 1940.87 776.22 1735.59 2328.26 2018.08 1617.94 798.84 2162.16 1500.48 998.30 2047.22 1330.47 816.25 1159.97 2783.04 1088.26 2705.90 2592.96 2224.66 2478.91 2848.27 3585.52 945.76 576.67 755.20

2505 2510 2515 2520 2525 2530 2535 m/z 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Relative Abundance

+12 Charge State

Spectrum 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

  • verlap of all variants

combined Clean spectrum from a single charge variant

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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 digest ZipChip HR Thermo 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
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Top down and bottom up proteomics

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Combined Top-Down and Bottom-Up Analysis in One Platform

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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

3.8

Time (min)

10 20 30 40 50 60 70 80 90 100

Relative Abundance

2.78 2.66 2.28 2.57 2.85 2.98 2.53 2.46 2.32 3.45 2.08 1.88

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

8.0

Time (min)

10 20 30 40 50 60 70 80 90 100

Relative Abundance

2.58 3.69 3.83 2.67 4.55 2.74 2.80 4.94 3.20 4.08 3.48

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

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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

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Available Resources

Brochure Spec sheets Posters TF.com page

  • Appl. Note
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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 BioPharma

  • ption 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/G2F 1.0 ppm 3.9 ppm 1.0 ppm 7.5 ppm 2.4 ppm 20 40 60 80 100 148056.78 148218.15 148379.38 147910.25 148542.25 147800 148200 148600 Mass Relative Intensity 1 2 3 Time (min) 100 100 Relative Abundance 100 0.96 2.35 2.51

a b c

Fully denatured Partially denatured

z=26 z=30 z=49 2000 4000 6000 8000 m/z 20 40 60 80 100 20 40 60 80 100 Relative Abundance 20 40 60 80 100 5695.4 5923.2 5484.6 5288.7 6183.7 3022.6 3085.5 3151.2 3444.2 4936.2 4777.1 5106.4 4627.8 3897.2 5288.8 3797.4

Native

5660 5700 5740 m/z 5695.4 5701.7 5707.7 5689.8 5714.0

a 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 Option CE separation was achieved on ZipChip HR

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Electropherogram of intact infliximab:

2 4 6 8

Time (min)

20 40 60 80 100

Relative Abundance

2.6 4.5 3.5

0 Lys 2 Lys 1 Lys

5700 5720 5740 m/z 50 100 50 100 Relative Abundance 50 100 5722.8 5717.9 5713.0

zoom z=26

Full MS spectra

5000 6000 m/z

50 100 50 100 Relative Abundance 50 100 5722.8 5510.8 5314.1 5951.7 5717.9 5506.2 5946.6 5309.5 5713.0 5941.4 5501.4 5305.0 z=26 z=25 z=27 z=28

RT 2.6 min RT 3.5 min RT 4.5 min

2 Lys 0 Lys 1 Lys

20 40 60 80 100 148767.8 148511.9 148929.2 148639.7 148673.3 148801.2 148835.7 149092.6 148964.9 148564.0 149031.6 149199.3 148311.6 149364.3 149481.5 148300 148500 148700 148900 149100 149300 149500 Mass

2 4 6 8

Time (min)

20 40 60 80 100

Relative Abundance

Sliding window deconvolution with RespectTM algorithm

Relative Intensity

[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 9.8 2 Lys G0F/G0F 148768.5 148767.8 4.8 2 Lys (G1F)2 or (G0F/G2F) 149092.8 149092.6 1.3 1 Lys G0F/G0F 148640.3 148639.7 4.3 1 Lys G0F/G1F 148802.5 148801.2 8.6 1 Lys (G1F)2 or (G0F/G2F) 148964.6 148964.9

  • 1.8

0 Lys G0F/G0F 148512.2 148511.9 1.6 0 Lys G0F/G1F 148674.3 148673.3 6.8 0 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
  • f 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 Option CE separation was achieved on ZipChip HR

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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 infliximab IdeS digested infliximab

Subunit Monoisotopic theo. mass Experimental mass  Mass (ppm) Light chain 23424.3946 23424.4671 3.1 Fd' 25631.5325 25631.5644 1.2 Fc/2 G0F 25316.5863 25316.6345 1.9 Fc/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

  • 4.8

Fc/2 G0F (2 SS bonds) 25312.5550 25312.5524 0.1 Fc/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 2.0 2.2 Time (min) 20 40 60 80 100 Relative Abundance 2.11 2.14 2.26

(Fc/2) -Lys F(ab’)2 Fc/2

120 k 15 k

z=44 z=45 z=42 z=43 2000 2500 m/z 20 40 60 80 100 Relative Abundance 2283.5 2231.7 2182.0 z=41 z=46 z=48

Deconvolution with Respect

98146.8

98000 100000 Mass

25312.5524 25184.4311 25474.5138 25346.4730

25100 25300 25500 Mass 20 40 60 80 100 Relative Intensity

(Fc/2 G0F) -Lys Fc/2 G0F (Fc/2 G1F) -Lys Fc/2 G1F

F(ab’)2

Deconvolution with Xtract

2.5 3.0 3.5 Time (min) 20 40 60 80 100 Relative Abundance 3.42 3.48 3.06

LC Fd’

(Fc/2) -Lys Fc/2

120 k

800 900 1000 1100 m/z 50 100 50 100 Relative Abundance 845.43 905.71 975.30 1014.31 1102.43 901.17 970.37 1009.23 1096.90 841.13

(Fc/2) -Lys Fc/2

LC Fd’

1000 1500 m/z 50 100 50 100 Relative Abundance 885.41 1026.87 1069.61 1166.81 938.54 1066.35 1172.93 1303.14 869.10 23424.4671 25631.5644

20 40 60 80 100 Relative Intensity Mass 25000 24000

Fd’ LC

(Fc/2 G0F) -Lys Fc/2 G0F (Fc/2 G1F) -Lys Fc/2 G1F 1200 1400 m/z 50 100 50 100 Relative Abundance 1327.2 1401.1 1261.0 1483.3 1201.0

(Fc/2) -Lys

1334.1 1267.4 1408.2 1207.0 1490.9

Fc/2

IdeS digest IdeS digest plus reduction

mAb Subunits Analysis in Protein mode

MS data was acquired on a QE HF-X with BioPharma Option CE separation was achieved on ZipChip HR

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Proprietary & Confidential

50 100

150931.7151091.8 152048.7 151888.7 151252.7 149974.5150134.3 153007.0 152210.5 150294.9 152845.5 153168.3 149176.3 149015.4 151314.3 153964.5 149336.8 154126.3 150358.5 152272.1 151411.8 152371.5 153802.9 150453.7 154921.9 148220.6 150580.1 153328.4 155143.3 151538.8 149499.1 154150.3 149559.6 148379.0 154758.8 155308.0 148871.3 154185.4 152497.4 154282.5 148440.6 151744.1 155805.4 153467.9 154661.2 155718.9 155474.7 154922.8

148500 150000 151500 153000 154500 156000 2.0 3.0 4.0 5.0 50 100 3.34 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 5500 50 100

5037.42 4874.89 5005.44 4905.77 4844.05 5069.29 5042.76 4911.01 4967.86 5074.72 5178.01 5205.51 4722.68 5101.28 4752.56 4813.01 5106.59 4782.45 5244.08 5145.03 5249.65 4692.75 5133.15 5271.44 5277.07 4995.22 5362.97 5391.49 5282.56 4608.56 5397.23 4637.63 5328.82 5304.57 4657.80 5431.31 5436.84 5471.36 5499.94

Electropherogram of Trastuzumab Emtansine MS Full Spectrum Deconvoluted Spectrum

M/Z

Relative Abundance

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 abundance DAR0 10.72 9.95 DAR1 7.45 33.47 DAR2 1.23 58.97 DAR3 2.37 79.90 DAR4 5.63 69.92 DAR5 9.08 49.14 DAR6 13.89 28.97 DAR7 9.93 12.64 DAR8 8.41 2.83

Average Drug‐to‐Antibody Ratio (DAR) is 3.47

MS data was acquired on a QE HF with BioPharma Option CE separation was achieved on ZipChip HR