life sciences poster summary - 908...

LIFE SCIENCES

OVERVIEWThe field of native mass spectrometry has advanced significantly over the past several years. We believe that the integration of a powerful online separation can advance the field of native mass spec even further. Capillary Electrophoresis (CE), coupled via ESI, is a natural fit for native mass spec analysis because it can be performed in native solvent conditions without concern for interactions with a chromatographic stationary phase. Additionally, the slow diffusion of large molecules is a benefit to electrophoretic separation efficiency, often enabling resolution between very minor structural differences of proteoforms. Successfully exploiting CE-ESI-MS for native analysis requires a level of optimization (of surface chemistry, channel geometry, solvent conditions, etc…) that has been beyond the reach of traditional CE-MS platforms. The continued advancement of our fully integrated microfluidic CE-ESI platform has enabled us to start tackling these extremely challenging applications. Here we demonstrate the unique capabilities of this approach; highlighting the high resolution separation of charge variants of a monoclonal antibody along with two different examples of native protein complex analysis.

METHODSInstrumentation. All work was performed using a commercially available microfluidic CE-ESI system (ZipChip®, 908 Devices Inc.). The microfluidic devices utilize a covalently attached, neutral polymer surface coating to prevent analyte interactions and suppress electroosmotic flow. For the monoclonal antibody analysis, a “high resolution native” (HRN) chip was used. This chip incorporates a 22 cm long separation channel and uses a new surface coating process to achieve high resolution protein separations under native conditions. The native complex separations were performed with a “high speed native” (HSN) chip. The shorter separation channel of the HSN device (10 cm), enables faster analysis of proteins and complexes under native conditions. The NIST mAb and blood samples were analyzed on a Thermo Exactive Plus EMR mass spectrometer. The beta galactosidase sample was analyzed on a Q Exactive UHMR mass spec.

Samples. 1. The NIST monoclonal antibody reference material was incubated at 45°C in 50 mM phosphate buffer at pH 8 to accelerate deamidation. Time points were collected at 0, 24, 48, 72, 96, and 120 hours. Samples were buffer exchanged into ZipChip Native Antibody BGE and diluted to a concentration of 0.25 mg/mL for analysis.

2. Whole human blood was diluted 20x with 10 mM ammonium acetate and centrifuged to remove particulates.

3. Beta galactosidase was prepared from a purified standard in 10 mM ammonium acetate to a concentration of 5 µM.

Data Processing. Data were visualized using Thermo Xcalibur QualBrowser . The NIST mAb data files were processed with an in house software program to accurately identify and assign relative quantitative abundance values to all of the observed species. All of the samples were run 3x to assign a standard deviation to each of the measurements.

POSTER SUMMARY Advancements in Native Analysis by Microchip Capillary Electrophoresis ESI-MS

Device Schematic27 mm

44 mm

Sample

Separation Channel

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120

ecnadnubA lanoitcarF

Hours of Incuba�on

No deamida�ons 1 deamida�on 2 deamida�ons 3 deamida�ons 4 deamida�ons

2K

0K

1K

Acidic0.25 mg/mL NIST mAbZipChip Native Antibody BGEHRN Chip1 nL injected500 V/cmExactive Plus EMR 2500 – 8000 m/z35,000 Resolution Setting3 microscans

Actual ZipChip

37 mm

54 mm

Full Spectra +26 Charge State Proteoforms Identified

C-term K Deamida�on Glycoform Theore�cal Mass Error (ppm) Rela�ve Abundance

0

0

G0F/G0F 148037.1 5.0 62.8G0F/G1F 148199.3 4.9 100.0G1F/G1F 148361.2 4.5 87.4G1F/G2F 148523.5 0.3 34.4G2F/G2F 148685.7 6.1 15.1

1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1

G0F/G0F 148166.3 1.7 0.8G0F/G1F 148328.4 0.8 1.1G1F/G1F 148490.4 16.1 0.9G1F/G2F 148652.7 2.9 0.7

20


1 G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2


44 mm

Sample

Separation Channel

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120




2K

0K

1K


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1 G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2


NATIVE ANTIBODY ANALYSIS WITH ZIPCHIP CE-ESI-MS


44 mm

Sample

Separation Channel

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120




2K

0K

1K


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1 G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2

Keeping the antibody in its native state during the microchip CE-ESI-MS analysis enables the high resolution separation of charge variants and the ability to assign accurate masses to each of the variant peaks. Zooming in on a single charge state of the mass spectra we see the excellent resolution between glycoform peaks achieved by running the orbitrap detection at a resolution setting of 35,000. We can also see how separation of variants before ESI removes the problem of spectral overlap and makes the mass spectrometer’s job easier. The table of proteoforms shows that we have confidently identified variations in C terminal lysine truncation, deamidation, and glycosylation for the NIST mAb. In total we have identified and assigned relative abundance values to 29 species spanning three orders of dynamic range in this single analysis. The key to extracting so much information lies in combining the high efficiency of the Native ZipChip separation with the high resolution and mass accuracy of the mass spectrometer.

C-term K Deamidation Glycoform Theoretical Mass Error (ppm) Relative Abundance

0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2

Proteoforms Identified


The base peak electropherogram for one of the forced deamidation time point samples (72 hours) shows the electrophoretic separation of both C-terminal lysine variants and deamidation variants. Deamidated variants can be seen for each of the three different lysine variants, and multiple deamidations can clearly be seen on the most intense, 0K variant. In addition, the single deamidation of the 0K variant reveals partial resolution of multiple peaks. These are likely explained by positional variants with the deamidation occurring at different amino acid sites.

The data were processed to assign fractional abundance to the observed species. Since this experiment was focused on deamidation, we’ve grouped all of the glycoforms and lysine variants together to indicate the total abundance of deamidated species relative to non-deamidated. A clear trend of increasing deamidation can be seen versus incubation time at pH 8 and 45°C. Species were detected with as many as four deamidations after 120 hours of incubation. The error bars indicate +/- 1 standard deviation for the 3 replicates of each sample run in this experiment.


44 mm

Sample

Separation Channel

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120




2K

0K

1K


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1 G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2


44 mm

Sample

Separation Channel

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120




2K

0K

1K


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1 G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2


44 mm

Sample

Separation Channel

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120




2K

0K

1K


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1 G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2


44 mm

Sample

Separation Channel

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120




2K

0K

1K


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1 G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2


Example 1: Native hemoglobin analyzed from raw human blood 64 kDa tetramer, pI ~7

Example 2: Beta galactosidase 466 kDa tetramer, pI ~5

Instrumentation. All work was performed using a commercially available microfluidic CE-ESI system (ZipChip, 908 Devices Inc.). The microfluidic devices utilize a covalently attached, neutral polymer surface coating to prevent analyte interactions and suppress electroosmotic flow. For the monoclonal antibody analysis, a “high resolution native” (HRN) chip was used. This chip incorporates a 22 cm long separation channel and uses a new surface coating process to achieve high resolution protein separations under native conditions. The native complex separations were performed with a “high speed native” (HSN) chip. The shorter separation channel of the HSN device (10 cm), enables faster analysis of proteins and complexes under native conditions. The NIST mAb and blood samples were analyzed on a Thermo Exactive Plus EMR mass spectrometer. The beta-galactosidase sample was analyzed on a Q-Exactive UHMR mass spec.

Samples. 1. The NIST monoclonal antibody reference material was incubated at

45°C in 50 mM phosphate buffer at pH 8 to accelerate deamidation. Time points were collected at 0, 24, 48, 72, 96, and 120 hours. Samples were buffer exchanged into ZipChip Native Antibody BGE and diluted to a concentration of 0.25 mg/mL for analysis.


3. Beta-galactosidase was prepared from a purified standard in 10 mM ammonium acetate to a concentration of 5 µM.

Data Processing. Data were visualized using Thermo XcaliburQualBrowser. The NIST mAb data files were processed with an in-house software program to accurately identify and assign relative quantitative abundance values to all of the observed species. All of the samples were run 3x to assign a standard deviation to each of the measurements.

The field of native mass spectrometry has advanced significantly over the past several years. We believe that the integration of a powerful online separation can advance the field of native mass spec even further. Capillary Electrophoresis (CE), coupled via ESI, is a natural fit for native mass spec analysis because it can be performed in native solvent conditions without concern for interactions with a chromatographic stationary phase. Additionally, the slow diffusion of large molecules is a benefit to electrophoretic separation efficiency, often enabling resolution between very minor structural differences of proteoforms. Successfully exploiting CE-ESI-MS for native analysis requires a level of optimization (of surface chemistry, channel geometry, solvent conditions, etc…) that has been beyond the reach of traditional CE-MS platforms. The continued advancement of our fully integrated microfluidic CE-ESI platform has enabled us to start tackling these extremely challenging applications. Here we demonstrate the unique capabilities of this approach; highlighting the high resolution separation of charge variants of a monoclonal antibody along with two different examples of native protein complex analysis.


Sample

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

Separation Channel

Overview

Methods

NIST mAb Forced Deamidation Time Point Analysis

The native antibody analysis method demonstrated above is now a robust and reproducible method that can be widely deployed for biotherapeutic characterization. This method is fast and efficient and, as shown here, it can be used to determine multiple critical quality attributes in one simple method with no sample prep. This method can be applied to a wide variety of mAbs, ADCs, and related molecules like bispecific mAbs and fusion proteins due to the relative similarity of all of these molecules.

The analysis of native protein complexes carries a significantly higher degree of difficulty due to the natural variability of molecular properties and the limitations placed on the composition of the BGE. We have demonstrated some of the potential for native protein complex analysis by microchip CE-ESI-MS with two different examples. The analysis of native hemoglobin from raw blood shows how an electrophoretic separation can produce clean mass spectra from “dirty” samples and also preserve the native structure of very fragile native protein complexes. The analysis of beta-galactosidase demonstrates the ability to reverse the electrophoretic field and separate species that are negatively charged in the native background electrolyte.

As a whole, the work presented here supports the notion that microchip CE-ESI-MS is uniquely well suited to native protein analysis. The highly developed native antibody method is an example of a mature method. Methods for proteins and protein complexes may require further optimization to achieve as high a level of performance, but the potential is clear.

We would like to thank Vicki Wysocki and her lab members for providing the beta-galactosidase sample and working with us to analyze the sample in their lab.

Conclusions and Acknowledgments

Native Antibody Analysis with ZipChip CE-ESI-MS

Keeping the antibody in its native state during the microchip CE-ESI-MS analysis enables the high resolution separation of charge variants and the ability to assign accurate masses to each of the variant peaks. Zooming in on a single charge state of the mass spectra we see the excellent resolution between glycoformpeaks achieved by running the orbitrap detection at a resolution setting of 35,000. We can also see how separation of variants before ESI removes the problem of spectral overlap and makes the mass spectrometer’s job easier. The table of proteoforms shows that we have confidently identified variations in C-terminal lysine truncation, deamidation, and glycosylation for the NIST mAb. In total we have identified and assigned relative abundance values to 29 species spanning three orders of dynamic range in this single analysis. The key to extracting so much information lies in combining the high efficiency of the Native ZipChip separation with the high resolution and mass accuracy of the mass spectrometer.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120

Frac

tiona

l Abu

ndan

ce

Hours of Incubation

No deamidations 1 deamidation 2 deamidations 3 deamidations 4 deamidations

2K

0K

1K

Acidic0.25 mg/mL NIST mAb1 nL injected (0.25 ng)ZipChip Native Antibody BGEHRN Chip500 V/cmExactive Plus EMR 2500 – 8000 m/z35,000 Resolution Setting3 microscans

The base peak electropherogram for one of the forced deamidation time point samples (72 hours) shows the electrophoretic separation of C-terminal lysine and deamidation variants. Deamidated variants can be seen for each of the three different lysine variants, and multiple deamidations can clearly be seen on the most intense, 0K variant. In addition, the single deamidation of the 0K variant reveals partial resolution of multiple peaks. These are likely explained by positional variants with the deamidation occurring at different amino acid sites.


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2

Advancements in Native Analysis by Microchip Capillary Electrophoresis-ESI-MS

Scott Mellors, Ashley Bell, Colin Gavin, and Erin Redman908 Devices, Inc., Boston, MA 02210

The technologies discussed in this poster are the subject of one or more granted/pending patents.

www.908devices.com/patents/

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

NL:3.79E7Base Peak

2000 4000 6000 8000 10000m/z

0

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

Dimer32 kDa

Hemoglobin Tetramer64 kDa

Whole blood diluted 20x with 10 mM ammonium acetate1.5 nL injectionPrototype Native BGE, pH 6.6ZipChip HSN +500 V/cmExactive Plus EMR

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

5 mM beta galactosidase1.5 nL injection (7.5 fmoles)Prototype Native BGE, pH 6.6ZipChip HSN-300 V/cmQE UHMR

4000 6000 8000 10000 12000 14000m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

466 kDatetramer

116 kDamonomer

Native Protein Complex Analysis

Example 1: Native hemoglobin analyzed from raw human blood64 kDa tetramer, pI ~7

Example 2: Beta-galactosidase466 kDa tetramer, pI ~5

0.25 mg/mL stressed NIST mAb1 nL injected (0.25 ng)ZipChip Native Antibody BGEHRN Chip500 V/cmExactive Plus EMR 2500 – 8000 m/z35,000 Resolution Setting3 microscans

Instrumentation. All work was performed using a commercially available microfluidic CE-ESI system (ZipChip, 908 Devices Inc.). The microfluidic devices utilize a covalently attached, neutral polymer surface coating to prevent analyte interactions and suppress electroosmotic flow. For the monoclonal antibody analysis, a “high resolution native” (HRN) chip was used. This chip incorporates a 22 cm long separation channel and uses a new surface coating process to achieve high resolution protein separations under native conditions. The native complex separations were performed with a “high speed native” (HSN) chip. The shorter separation channel of the HSN device (10 cm), enables faster analysis of proteins and complexes under native conditions. The NIST mAb and blood samples were analyzed on a Thermo Exactive Plus EMR mass spectrometer. The beta-galactosidase sample was analyzed on a Q-Exactive UHMR mass spec.

Samples. 1. The NIST monoclonal antibody reference material was incubated at

45°C in 50 mM phosphate buffer at pH 8 to accelerate deamidation. Time points were collected at 0, 24, 48, 72, 96, and 120 hours. Samples were buffer exchanged into ZipChip Native Antibody BGE and diluted to a concentration of 0.25 mg/mL for analysis.


3. Beta-galactosidase was prepared from a purified standard in 10 mM ammonium acetate to a concentration of 5 µM.

Data Processing. Data were visualized using Thermo XcaliburQualBrowser. The NIST mAb data files were processed with an in-house software program to accurately identify and assign relative quantitative abundance values to all of the observed species. All of the samples were run 3x to assign a standard deviation to each of the measurements.

The field of native mass spectrometry has advanced significantly over the past several years. We believe that the integration of a powerful online separation can advance the field of native mass spec even further. Capillary Electrophoresis (CE), coupled via ESI, is a natural fit for native mass spec analysis because it can be performed in native solvent conditions without concern for interactions with a chromatographic stationary phase. Additionally, the slow diffusion of large molecules is a benefit to electrophoretic separation efficiency, often enabling resolution between very minor structural differences of proteoforms. Successfully exploiting CE-ESI-MS for native analysis requires a level of optimization (of surface chemistry, channel geometry, solvent conditions, etc…) that has been beyond the reach of traditional CE-MS platforms. The continued advancement of our fully integrated microfluidic CE-ESI platform has enabled us to start tackling these extremely challenging applications. Here we demonstrate the unique capabilities of this approach; highlighting the high resolution separation of charge variants of a monoclonal antibody along with two different examples of native protein complex analysis.


Sample

ESI Corner

MS Inlet

+15 kV

+2 kV

Waste

BGE

BGE

Separation Channel

Overview

Methods

NIST mAb Forced Deamidation Time Point Analysis

The native antibody analysis method demonstrated above is now a robust and reproducible method that can be widely deployed for biotherapeutic characterization. This method is fast and efficient and, as shown here, it can be used to determine multiple critical quality attributes in one simple method with no sample prep. This method can be applied to a wide variety of mAbs, ADCs, and related molecules like bispecific mAbs and fusion proteins due to the relative similarity of all of these molecules.

The analysis of native protein complexes carries a significantly higher degree of difficulty due to the natural variability of molecular properties and the limitations placed on the composition of the BGE. We have demonstrated some of the potential for native protein complex analysis by microchip CE-ESI-MS with two different examples. The analysis of native hemoglobin from raw blood shows how an electrophoretic separation can produce clean mass spectra from “dirty” samples and also preserve the native structure of very fragile native protein complexes. The analysis of beta-galactosidase demonstrates the ability to reverse the electrophoretic field and separate species that are negatively charged in the native background electrolyte.


We would like to thank Vicki Wysocki and her lab members for providing the beta-galactosidase sample and working with us to analyze the sample in their lab.

Conclusions and Acknowledgments

Native Antibody Analysis with ZipChip CE-ESI-MS

Keeping the antibody in its native state during the microchip CE-ESI-MS analysis enables the high resolution separation of charge variants and the ability to assign accurate masses to each of the variant peaks. Zooming in on a single charge state of the mass spectra we see the excellent resolution between glycoformpeaks achieved by running the orbitrap detection at a resolution setting of 35,000. We can also see how separation of variants before ESI removes the problem of spectral overlap and makes the mass spectrometer’s job easier. The table of proteoforms shows that we have confidently identified variations in C-terminal lysine truncation, deamidation, and glycosylation for the NIST mAb. In total we have identified and assigned relative abundance values to 29 species spanning three orders of dynamic range in this single analysis. The key to extracting so much information lies in combining the high efficiency of the Native ZipChip separation with the high resolution and mass accuracy of the mass spectrometer.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 24 48 72 96 120

Frac

tiona

l Abu

ndan

ce

Hours of Incubation

No deamidations 1 deamidation 2 deamidations 3 deamidations 4 deamidations

2K

0K

1K

Acidic0.25 mg/mL NIST mAb1 nL injected (0.25 ng)ZipChip Native Antibody BGEHRN Chip500 V/cmExactive Plus EMR 2500 – 8000 m/z35,000 Resolution Setting3 microscans

The base peak electropherogram for one of the forced deamidation time point samples (72 hours) shows the electrophoretic separation of C-terminal lysine and deamidation variants. Deamidated variants can be seen for each of the three different lysine variants, and multiple deamidations can clearly be seen on the most intense, 0K variant. In addition, the single deamidation of the 0K variant reveals partial resolution of multiple peaks. These are likely explained by positional variants with the deamidation occurring at different amino acid sites.


Actual ZipChip

37 mm

54 mm



0

0


1


2G0F/G0F 148039.1 4.6 0.2G0F/G1F 148201.3 9.6 0.6G1F/G2F 148525.5 0.3 0.2

1

0


1


20


1G0F/G0F 148294.5 16.9 0.1G1F/G1F 148618.5 22.4 0.2

Advancements in Native Analysis by Microchip Capillary Electrophoresis-ESI-MS

Scott Mellors, Ashley Bell, Colin Gavin, and Erin Redman908 Devices, Inc., Boston, MA 02210

The technologies discussed in this poster are the subject of one or more granted/pending patents.

www.908devices.com/patents/

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

NL:3.79E7Base Peak

2000 4000 6000 8000 10000m/z

0

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

Dimer32 kDa

Hemoglobin Tetramer64 kDa

Whole blood diluted 20x with 10 mM ammonium acetate1.5 nL injectionPrototype Native BGE, pH 6.6ZipChip HSN +500 V/cmExactive Plus EMR

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

5 mM beta galactosidase1.5 nL injection (7.5 fmoles)Prototype Native BGE, pH 6.6ZipChip HSN-300 V/cmQE UHMR

4000 6000 8000 10000 12000 14000m/z

0

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

466 kDatetramer

116 kDamonomer

Native Protein Complex Analysis

Example 1: Native hemoglobin analyzed from raw human blood64 kDa tetramer, pI ~7

Example 2: Beta-galactosidase466 kDa tetramer, pI ~5

0.25 mg/mL stressed NIST mAb1 nL injected (0.25 ng)ZipChip Native Antibody BGEHRN Chip500 V/cmExactive Plus EMR 2500 – 8000 m/z35,000 Resolution Setting3 microscans

NATIVE PROTEIN COMPLEX ANALYSIS


ZipChip and ZipChip Interface hardware/consumable kits are for research use only, and cannot be used for diagnostic purposes.

_______________________________________________

908 Devices+1.857.254.1500 | [email protected] www.908devices.com ZipChip is subject to export controls including those of the Export Administration Regulations of the U.S. Department of Commerce, which may restrict or require licenses for the export of product from the United States and their re-export to and from other countries. Patented technology www.908devices/patents © 2019 908 Devices.

CONCLUSIONS AND ACKNOWLEDGMENTSThe native antibody analysis method demonstrated above is now a robust and reproducible method that can be widely deployed for biotherapeutic characterization. This method is fast and efficient and, as shown here, it can be used to determine multiple critical quality attributes in one simple method with no sample prep. This method can be applied to a wide variety of mAbs , ADCs, and related molecules like bispecific mAbs and fusion proteins due to the relative similarity of all of these molecules.

The analysis of native protein complexes carries a significantly higher degree of difficulty due to the natural variability of molecular properties and the limitations placed on the composition of the BGE. We have demonstrated some of the potential for native protein complex analysis by microchip CE-ESI-MS with two different examples. The analysis of native hemoglobin from raw blood shows how an electrophoretic separation can produce clean mass spectra from “dirty” samples and also preserve the native structure of very fragile native protein complexes. The analysis of beta galactosidase demonstrates the ability to reverse the electrophoretic field and separate species that are negatively charged in the native background electrolyte.


We would like to thank Vicki Wysocki and her lab members for providing the beta galactosidase sample and working with us to analyze the sample in their lab.

Authors:Scott Mellors, Ashley Bell, Colin Gavin, and Erin Redman908 Devices, Inc., Boston, MA

life sciences poster summary - 908...

Documents