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Innovation with IntegrityBioPharma

mAb Development made easy

maXis II for Biopharma Analysis

Determining the monoisotopic mass for large molecules (> 25 kDa) and related variants is essential for mAb developability assessments during early stage development.

The maXis II UHR-QTOF mass spectrometer together with the SNAP-II deconvolution algorithm possesses an intrinsic edge for the rapid profiling of large proteins and their heterogeneities.

The maXis II acquires True Isotopic Pattern (TIP) raw data that allows for rapid determination of modifications such as deamidation of the light chain (LC). Coupled to SNAP-II algorithm, these subtle modifications can be routinely characterized at the sub-unit level.

Sub-unit characterization (e.g. de-amidation) using the power of True Isotopic Pattern (TIP) raw data

maXis II: Unmatched Performance

True Isotopic Pattern (TIP)

23435 23440 23445 23450 23455

L Chain

Asn 30

MWth = 23428.524MWob = 23428.530

Asp30 orisoAsp30

Asn 30

MWth = 23429.508MWob = 23429.522∆mass = +0.992Da

Asp30 orisoAsp30

The ‘de facto’ standard for biologics

Intact Analysis

MaxEnt deconvolution spectrum measured in blue. Simulated mass spectrum in black.

Sub-unit Analysis

F(ab')2

Fc/2

Released Glycan Analysis

Glycopeptide Analysis

Higher Order Analysis

Increase speed to clinic with the maXis II:

The maXis II comes with electron transfer dissociation (ETD) capability as well as a high-mass option (HMO) for native MS applications.

• Understand and predict PK properties of antibodies by thorough characterization of glycans• Accurately determine deamidation• Rapid drug-antibody ratio (DAR) analysis• Reduce downstream manufacturing risks• Monitor batch to batch variability

Mass spectrum of a heavy chain with resolved isotopes

~80,000 resolution

Mr mono (meas) = 50613.041 DaMr mono (expected) = 50613.004 Da

∆Mr/Mr = 0.73 ppm

50625 50630 50635 50640 50645 50650 50655 50660 50665 m/z

Glycosylation is a crucial quality attribute for many biopharmaceuticals, as small changes in glycosylation profiles can have significant impact on stability, efficacy and/or safety.

Identifying, monitoring and controlling the glycosylation levels is key for successful biopharmaceutical development and the maXis II confidently addresses these key challenges in many pharmaceutical labs today at the intact, subunit, glycopeptide and released glycan level.

Superior Glycan Profiling Performance

Confidently determine:• Relative abundances (The most glycan IDs and

1-sigma groups in NISTmAb round robin study)*

• Composition (glycopeptide or released)**

• Localization within protein sequence

(glycopeptide)

Profiling at the intact level usually has a mass accuracy of around 10 ppm accuracy, as illustrated above. With resolved isotopes (< 50 kDa) it drops under 1 ppm. The maXis II UHR-QTOF sets the performance standard, with an unrivaled dynamic range for large proteins and glycoforms.

146500 146600 146600 146800 146900 147000 m/z

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Inte

nsi

ty

x103

1465

93.8

58G

2F/a

gly

con

1467

54.8

17G

0F/a

gly

con

1469

18.6

84G

2F/a

gly

con

146900 147400 147900 148400 148900 m/z

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

Inte

nsi

ty

x104

1465

93.8

58G

0F/a

gly

con

1476

28.7

60G

0F/G

0F-G

lcN

Ac

(1) MaxEnt Spectrum

1467

54.8

17G

1F/a

gly

con

1465

93.8

58G

2F/a

gly

con

1478

36.1

48

G0F

/G0F

-Glc

NA

c

1480

38.5

46G

0F/G

0F

1479

98.8

76G

0F/G

1F-G

lcN

Ac

1481

63.5

35G

0F/G

0F +

K14

8200

.633

G0F

/G1F

1483

62.8

59G

1F/G

1F

1483

27.3

77G

0F/G

1F +

K

1485

25.1

37G

1F/G

2F

1484

89.7

36G

1F/G

1F +

K

1485

25.1

37G

2F/G

2F

1488

48.

683

G2F

/G2F

+ H

ex

*Maria Lorna A. De Leoz et al: NIST Interlaboratory Study on the Glycosylation of NISTmAb, NIST Pubs(2017), DOI: 10.6028/NIST.IR.8186

**H. Hinneburg et al.: The Art of Glycopeptide Destruction, J. Am. Soc. Mass Spectrom. (2015), DOI: 10.1007/s13361-015-1308-6

Increased Confidence in Released Glycan Analysis• Empirical spectra library comprising accurate mass and MS/MS

spectra of 2AB and RapiFluor® labeled glycans (25 glycoproteins

including most IgG subtypes and over 15 cell lines)

• Optimized LC and MS acquisition method

• Data processing with built-in expert knowledge. Scoring based on

fragment intensities and quantitation of coeluting glycans

• Report generation including MS and fluorescence based data with

minimized user intervention

• Workflow can be applied to regulated environments

GlycoFilerTM ResultsSample Preparation

2-aminobenzoic acid

RapiFluor

Compound ID

20

Main peaksassigned withglycan structures

GlycoFilerTM Workflow

• Enzymatic release of glycans from

the protein

• Fluorescence labeling

• HILIC-UPLC-FLR-CID-MS/MS

MeasurementHILIC-UPLC-FLR-CID-MS-MS

FLR Detection MS DetectionESI-QTOF-MS/MS

Data ProcessingAutomated

Integrationof FLR Peaks

IdentificationMatch with Glycan

Spectra Library

FLR-MS Data Hybridization

Table of glycan structures correlated to relative fluorescence peak areas

Data ProcessingAutomated

Reporting

Certificate of Analysis

Calculation of biologically relevant parameters

Dr. Sven Bahrke, Glycotype GmbH, Germany

“ GlycoFiler TM improved our lab productivity by reducing the time spent on evaluating data, especially in the case of complex glycan mixtures with coeluting peaks. In addition the ability to unambigu-ously identify and precisely quantify even low abundant structures reduces the non-annotated peak area to a minimum as well as the risk of false identifications. Integrated and automated quality control of the analyses as well as straightforward comparison of results between products round out the GlycoFiler TM workflow.“

EGFR (8 glycosylation sites)

Extract from GlycoFiler reports: Fluorescence chromatograms for an IgG1 and EGFR released glycans annotated with the main identified structures.

The maXis II easily detects and quantifies micro-heterogeneities directly on intact antibodies including under native conditions. This makes the maXis II the perfect platform to rapidly develop assays for drug distribution and average drug loading (DAR) without waiting for HPLC methods to be established.

Comparability with orthogonal methods and robustness makes it possible to validate these methods for GLP operations.

Rapidly Characterize Drug-Antibody Conjugates under GLP

• Interday• Intersite• Under GLP

Validation of drug distribution and DAR assay under native conditions on the maXis II UHR-QTOF

Method Reproducibility Assessment

%Peak Area

Prep 1Prep 2Prep 3Prep 4Prep 5Prep6

MeanSD

%RSD

0.0000.0000.0000.0000.0000.0000.0000.0000.000

0-Drug% 1-Drug% 2-Drug% DAR

3.8893.6194.1774.1993.8204.1923.9830.2065.162

96.11196.38195.82395.80196.18095.80896.0170.2060.214

1.961.961.961.961.961.961.960.000.00

%Peak AreaAnalyst

Analyst 1 - Day 1

Analyst 2 - Day 1

Analyst 1 - Day 2

Mean

SD

RSD

% 0-Drug

% 1-Drug

% 2-Drug DAR

0.000

0.000

0.000

0.000

0.000

0.000

4.177

3.500

3.883

3.853

0.277

7.193

95.823

96.500

96.117

96.147

0.277

0.288

1.96

1.96

1.96

1.96

0.00

0.00

Top-Down sequencing using ETD

One software platform including features such as intact protein and sub-unit analysis or peptide screening and quantitation workflows, which routinely provide artifact free quantification with highest dynamic range, fully integrating LC-UV, MS and MS/MS data.

Integrated data acquisition. Simple GUI for starting and monitoring acquisition runs.

Predefined, customizable report formats, supports 21 CFR Part 11 requirements.

BioPharma Compass: Designed in collaboration with the pharmaceutical industry

Automatic result assessment, • Algorithm based similarity scoring

Butterfly plots for easy comparison of chromatograms and spectra

Released glycans and glycopeptides MS/MS based identification

Protein Peaks Expected Peaks Confirmed Cosine Similarity Accepted

LC 2 2 1.0000 Yes

Fc2 9 9 1.0000 Yes

Fd 2 2 1.0000 Yes

BioPharma Compass

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Bruker Daltonik GmbH

Bremen · GermanyPhone +49 (0)421-2205-0

Bruker Scientific LLC

Billerica, MA · USA Phone +1 (978) 663-3660

ms.sales.bdal@bruker.com - www.bruker.com

For research use only. Not for use in diagnostic procedures.

Scan the QR-Code for more Details

7 Reasons the maXis II makes mAb development easy

• 80,000 FSR (Full Sensitivity Resolution) with high intra-scan dynamic range• True Isotopic Pattern (TIP) not limited by space charge effects for accurate DAR analysis• SNAP-II algorithm for accurate monoisotopic mass determination for large biologics• Supports 21 CFR Part 11• Electron Transfer Dissociation• Native mass spectrometry enabled• Glycan analysis with Glycofiler library