monitoring released n-glycans in biopharma development/qc with fluorescence & mass detection

27
©2015 Waters Corporation 1 GlycoWorks RapiFluor-MS and the ACQUITY QDa Mass Detector A fast and powerful new solution for released N-glycan monitoring

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Page 1: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 1

GlycoWorks RapiFluor-MS

and the

ACQUITY QDa Mass Detector

A fast and powerful new solution

for released N-glycan monitoring

Page 2: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 2

Presentation Outline

Market feedback: Unmet needs in biopharma

GlycoWorks™ RapiFluor-MS™ N-Glycan Kit

and the ACQUITY® QDa® Detector

Powerful N-glycan profiling with mass confirmation

Greater speed, confidence and productivity

Reimagine glycan analysis

Page 3: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 3

Market feedback

“We need a faster, more robust method for doing routine glycan

analysis. Our current sample prep method takes a day and a

half, and our results are not as sensitive, or as clear cut as we’d

like them to be.”

“It’s critical that we monitor the glycan profile of our protein

therapeutic and ensure that it remains consistent as we move

through development, production and final release.”

“Sometimes we see unexpected peaks or shoulders in our

fluorescent trace and the only way to obtain mass data is to

send samples to our core mass spec lab.”

The bottom line: Biopharmaceutical laboratoriesneed a faster workflow with improved selectivity and

sensitivity for routine glycan profiling.

Page 4: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 4

AccQ•Fluor

RapidFluorescence

Patent Pending

RapiFluor-MS

RapiFluor-MS ReagentBuilt on Waters’ Biopharm Applications Knowledge

From Waters’ expertise in rapid,

fluorescence labeling of amino acids

Enhanced chemical properties

for glycan analysis:

Rapid tagging

Efficient fluorescence

Enhanced ionization efficiency

Page 5: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 5

GlycoWorks RapiFluor-MS N-Glycan KitAt least 16x faster than current 2AB methods

New proprietary reagent mix reduces de-glycosylation time from 16+ hours down to 15 minutes!

Novel label reduces labeling time to just 5 minutes, and dramatically improves FLR and MS sensitivity

Samples ready for analysis in 30 minutes. Can also be dried down for later analysis

>1 work day

Conventional 2AB Method

Pre-Labeling, Clean-Up + Drying

3 hours

Drying

2 hours

Deglycosolation

16+ hours

Labeling

3 hours

Clean-Up

0.1 hours

RapiFluor-MSMethod

30 Minutes!

15 min

5 min

10 min

Page 6: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 6

Unparalleled FLR and MS Sensitivity

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Fluorescence MS (BPI)

Instant AB Labeled

RapiFluor-MS Labeled

2-AB Labeled

Rela

tive P

erf

orm

ance (

%)

52.5

7.0

0.1 0.6

Procainamide Labeled

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30.0*

7.0*

RapiFluor-MS vs Instant: 2x ↑ in FLR and 1000x ↑ in MSRapiFluor-MS vs Traditional: 14x ↑ in FLR and 160x ↑ in MSRapiFluor-MS vs PCA: 14x ↑ in FLR and 3x ↑ in MS

Page 7: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 7

FA2BG2S2

How RapiFluor-MS Helps…Higher Charge for MS Compatibility

5.3e2

m/z500 1000 1500 2000

%

0

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m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6

2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4

2AB_hIgG_halfload_20Nov14_10 495 (8.689)529

RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5

m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6

2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4

2AB_hIgG_halfload_20Nov14_10 495 (8.689)529

RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5

m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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m/z500 1000 1500 2000

%

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RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6

2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4

2AB_hIgG_halfload_20Nov14_10 495 (8.689)529

RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5

m/z500 1000 1500 2000

%

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RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6

2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4

2AB_hIgG_halfload_20Nov14_10 495 (8.689)529

RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5

500 2000 m/z

500 2000 m/z

500 2000 m/z

500 2000 m/z

2.9e4

1.2e5

3.5e62+

3+

2+ 2+

1+

FA2

2-ABRapiFluor-MS

Example courtesy of M.Lauber

Page 8: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 8

The ACQUITY QDa Mass Detector A pioneering product with mass appeal

Innovative design focused on ease of

use for chromatographers

Empowering analytical chemists

everywhere with orthogonal mass

detection – added information with

every sample

Compact, robust and affordable:

Built for constant use with a wide

variety of chromatographic conditions

Seamlessly integrates with

Empower®-based UPLC® Systems for

ease-of-use and GMP compliance

www.waters.com/qda

Page 9: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 9

Easily Added to Existing Systems

Existing stack

Easy to deploy

Fully integrated with Empower CDS Software

Minimal training required

110/220V operation

Minimal maintenance

ACQUITY QDa

Existing stack +1upgrade

Page 10: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 10

Familiar Graphical User Interface for Ease-Of-Use and Fast Adoption

Empower GUI is identical to that of a FLR for setup, data viewing and reporting Very little training needed

Quick addendum to current lab SOP

Page 11: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 11

Automated Start Up ProvidesRobust, Reproducible Performance

Automated resolution and calibration occurs with each

start-up ensuring mass information is accurate and precise

ESI interface optimized for UPLC performance to ensure

chromatographic resolution, sensitivity and throughput is

preserved

Graphical ACQUITY QDamonitor display Enables easy viewing and adjustment of system parameters

Page 12: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 12

Disposable Sample Aperture and Capillary for Easy Maintenance

Sample Aperture:

As simple as replacing a detector lamp

Capillary:

No cutting or assembly required

Page 13: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 13

ACQUITY QDa in Summary

A pioneering product that is empowering analytical chemists

and chromatographers everywhere with the power of mass

detection

Brings greater insight into every peak, enhanced monitoring

and streamlined workflows for improved productivity

Easy to deploy, simple to use and maintain, compact, robust

and affordable!

www.waters.com/qdabiopharm

Page 14: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 14

A Powerful Released N-Glycan Monitoring Solution

What the following data demonstrates:

The ACQUITY QDa Detector detects a broad range of

RapiFluor-MS labeled N-glycans across a large dynamic range

The ACQUITY QDa seamlessly integrates with UPLC workflows

using Empower Software, enabling GMP-compliant reporting

with mass data

The ACQUITY QDa and RapiFluor-MS combination enables

faster methods and higher throughput N-glycan profiling for

improved process development, optimization and monitoring

Page 15: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 15

Routine N-Glycan Detection with Matching FLR and MS response

Minutes5 10 15 20 25 30 35 40

Minutes

5 10 15 20 25 30 35 40

Detection across a broad range of glycoforms:

IgGSimple bi-antennary structures

RNase BHigh mannose structures

FetuinLarge, complex and highly sialylatedstructures

FLR

ACQUITYQDa

Page 16: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 16

Routine N-Glycan Detection with Matching FLR and MS response

Minutes5 10 15 20 25 30 35 40

Minutes5 10 15 20 25 30 35 40

EU

0

20

40

60

FLR

Inte

nsity

1x106

2x106

3x106

4x106

5x106

6x106

TIC

Key Takeaway:Equivalent FLR and MS response all within a single run, with the added benefit of mass confirmation.

Page 17: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 17

IgG Glycan Profile and Structure Confirmation Using the ACQUITY QDa

Key Takeaway: Large dynamic range – can clearly see most abundant and least abundant glycoforms.

0.00

240.00

480.00

In

ten

sit

y

0.0

1.6x106

3.2x106

Minutes10.00 15.00 20.00 25.00 30.00

896.0

Inte

nsity

0.0

12000.0

24000.0

m/z600 1200

A2G1b0.5% RPA

888.2

Inte

nsity

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300000

600000

m/z600 1200

FA220.5% RPA

EU

Page 18: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 18

Ions Detected with ACQUITY QDafor IgG MS Standard

Glycan Mavg (Da) [M+H]+1 [M+2H]+2 [M+3H]+3

A2 1628.6 1629.6 815.3 543.9

FA2 1774.7 1775.7 888.4 592.6

FA2B 1977.9 1978.9 990.0 660.3

A2G1a 1790.7 1791.7 896.4 597.9

A2G1b 1790.7 1791.7 896.4 597.9

FA2G1a 1936.9 1937.9 969.4 646.6

FA2G1b 1936.9 1937.9 969.4 646.6

FA2BG1a 2140.1 2141.1 1071.0 714.4

FA2BG1b 2140.1 2141.1 1071.0 714.4

A2G2 1952.9 1953.9 977.4 652.0

FA2G2 2099.0 2100.0 1050.5 700.7

FA2BG2 2302.2 2303.2 1152.1 768.4

FA2G1S1 2228.1 2229.1 1115.1 743.7

FA2G2S1 2390.3 2391.3 1196.1 797.8

FA2BG2S1 2593.5 2594.5 1297.7 865.5

FA2G2S2 2681.5 2682.5 1341.8 894.8

FA2BG2S2 2884.7 2885.7 1443.4 962.6

Page 19: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 19

How About Something More Complex?

EU

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FLR

Bovine Fetuin RapiFluor-MS Labeled N-Glycans (~30 pmol)

Key Takeaway:Demonstrates the upper limit of detection, and the ability to detect large, complex multi-antennary structures

Page 20: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 20

Largest Fetuin Structure Appears in the ACQUITY QDa Mass Window

QDa Positive Scan

Apex

1161.78

1167.43

1169.09

1174.72

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A3G3S4[M+3H]3+

A complex tri-antennary tetra-sialylatedglycoform

Analysis on our Xevo® G2-XS QTofconfirms the +3 charge state is the only charge state for this glycoform

Page 21: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 21

Empower Software Integration:Easily Combine Optical and Mass Data

Empower integration enables annotation of peaks with both names and m/z data

A2 -

815.1

FA2 -

888.2

FA2B -

989.6

A2G

1a -

896.2

A2G

1b -

896.0

FA2G

1a -

969.3

FA2G

1b -

969.2

FA2BG

1a -

1070.7

FA2BG

1b -

1070.6

A2G

2 -

977.1

FA2G

2 -

1050.3

FA2BG

2 -

1151.8

FA2G

1S1 -

1114.6

FA2G

2S1 -

1196.0

FA2BG

2S1 -

865.3

FA2G

2S2 -

894.7

FA2BG

2S2 -

962.2

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Page 22: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 22

Empower Software Integration:GMP Compliant Reporting with Mass

>

Page 23: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 23

Developing a Rapid Method for Released N-Glycan Analysis

EU

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Method

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Trastuzumab N-Glycan Analysis

Page 24: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 24

Enabling Accelerated Bioprocess Development

EU

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SIR Overlay

Trastuzumab N-Glycan AnalysisRapiFluor-MS labeled glycans - 10 minute method In

tensity

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A2G(4)1896.4 m/z

F(6)A2888.4 m/z

F(6)A2G(4)1969.4 m/z

F(6)A2G(4)21050.5 m/z

A2815.3 m/z

F(6)A2G(4)2S1865.5 m/z

M5774.1 m/z

Page 25: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 25

Monitoring Key Glycan Ratios:Keeping Tabs on Mannose 5

Time 1 Time 2 Time 3

Low Medium High

Mean 0.58 0.91 1.16

StDev 0.03 0.05 0.04

% RSD 5.44 5.47 3.85

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F(6)A2G(4)1SIR: 969.4 m/z

M5SIR: 774.1 m/z

Man5: F(6)A2G(4)1 Ratio

Page 26: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 26

GlycoWorks RapiFluor-MS

N-Glycan Kit enables at least 16X

faster sample prep time

RapiFluor-MS labeled N-glycans

are well observed by the

ACQUITY QDa mass detector

Empower Software supports GMP

compliant workflows

The ACQUITY QDa Detector

enables greater selectivity, faster

methods and mass confirmation

for increased confidence and

productivity

In Summary

Fast and powerful released N-glycan monitoring with the confidence of mass confirmation

RapiFluor-MS+

ACQUITY QDa

www.waters.com/qdabiopharm

Page 27: Monitoring Released N-Glycans in Biopharma Development/QC with Fluorescence & Mass Detection

©2015 Waters Corporation 27

ACQUITY QDA Detector for Biopharm: www.waters.com/qdabiopharm

Reimagine glycan analysis: www.waters.com/glycans