andrew williamson applications specialist presentation.pdf · 7 0.75 133.0976 ornithine 8 0.78...

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




What is the ZipChip System?

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* ZipChip system is sold exclusively by Thermo Fisher in Europe and APAC


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



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


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


Common Applications Performed on ZipChip-MS Platform

Metabolomics

Intact mAb/protein, and ADC characterization

mAb subunit analyses

Glycomics and glycoproteomics

Peptide mapping


How ZipChip works


• 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




• 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




• 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


Anatomy of ZipChip

MS Inlet


Glass microfluidic chip in PEEK housing

+HV1

+HV2

Well 1Waste

Well 2BGE

Well 3Sample

Well 4BGE

Well 2BGE

Well 3Sample

Well 1Waste

Well 4BGE


+HV1

+HV2

Well 1Waste

Well 2BGE

Well 3Sample

Well 4BGE

Sample Injection

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


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

‐‐++


Small Molecule Analysis/ Metabolomics


ZipChip Separation of Amino Acids

2.5 M Promega Complete AA MixZipChip HSZipChip Metabolite KitExactive EMRField Strength: 1000 V/cm

1.8min


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


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


Biopharmaceuticals


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

0

20

40

60

80

1000

20

40

60

80

100

Rel

ative

Abu

ndan

ce

0

20

40

60

80

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)

20

40

60

80

100

Rel

ativ

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bund

ance

2.55

2.52

2.49

0

0 Lys

1 Lys

2 Lys

4600 4800 5000 5200 5400m/z

0

50

1000

50

100

Rel

ative

Abun

danc

e 0

50

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


ZipChip Analysis of NIST Human Plasma, SRM 1950

0

50

100

150

200

250

300

350

Concen

tration (m

icroMolar)

Highly Accurate Quantitation

ZipChip NIST Reference Values

* * * * *

* No NIST Reference Value


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

ativ

e A

bund

ance

Time (Min)

Electropherogram of Digested NIST mAb



Glycoproteomics


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)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

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0

10

20

30

40

50

60

70

80

90

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

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

1000

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

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

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

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ance

+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


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


Top down and bottom up proteomics


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


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)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

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)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

eAb

unda

nce

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


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


Available Resources

Brochure

Spec sheets

Posters

TF.com page

Appl. Note


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

40

60

80

100 148056.78

148218.15

148379.38

147910.25 148542.25147800 148200 148600

Mass

Rel

ativ

eIn

tens

ity

1 2 3Time (min)

0

1000

100

Rel

ativ

e A

bund

ance

0

100 0.96

2.35

2.51

abc

Fully denatured

Partially denatured

z=26

z=30

z=49

2000 4000 6000 8000m/z0

20406080

1000

20406080

100

Rel

ativ

e A

bund

ance

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


Electropherogram of intact infliximab:

0 2 4 6 8Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

2.6

4.53.5

0 Lys2 Lys1 Lys

5700 5720 5740m/z

050

1000

50100

Rel

ativ

e A

bund

ance

050

100 5722.8

5717.9

5713.0

zoom

z=26Full MS spectra

5000 6000m/z

050

1000

50

100

Rel

ativ

e A

bund

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

60

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

ativ

e Ab

unda

nce Sliding window deconvolution

with RespectTM algorithm

Rel

ativ

e In

tens

ity

[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



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

2.0 2.2 Time (min)0

20

40

60

80

100

Rel

ativ

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bund

ance

2.11

2.14

2.26

(Fc/2) -LysF(ab’)2

Fc/2

120 k 15 k

z=44

z=45z=42

z=43

2000 2500m/z0

20

40

60

80

100

Rel

ativ

e A

bund

ance

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

100

Rel

ativ

e In

tens

ity (Fc/2 G0F) -LysFc/2 G0F

(Fc/2 G1F) -LysFc/2 G1F

F(ab’)2

Deconvolutionwith Xtract

2.5 3.0 3.5 Time (min)0

20

40

60

80

100

Rel

ativ

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bund

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3.42 3.48

3.06

LC

Fd’(Fc/2) -LysFc/2

120 k

800 900 1000 1100m/z0

50

1000

50

100

Rel

ativ

e A

bund

ance

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

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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



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


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