Innovation with IntegrityMALDI-TOF/TOF MS

The enhanced Fast Results for Efficient Biopharmaceutical Research

ultrafle treme

Know more aboutyour samples – faster!

The next generation, enhanced ultrafleXtreme™ com-prises the most recent MALDI-TOF/TOF technology based on the market leading ultrafleXtreme series.Expand your expectations and explore what you can achieve with the most modern, industrial standard MALDI-TOF/TOF system.

Applications such as in-depth characterization of biopharmaceuticals and tissue imaging benefit from a multitude of technology innovations and solutions.

Biopharmaceuticals Character- ization - unique speed, quality and simplicity

Glycoprotein Analysis – the next life science frontier at your fingertips

QC Applications - fill and finish or clone selection - fast results for more efficient processes

Solution provider for any challenging sample

This is where MALDI truly accelerates your research

QC and MALDI assays with unmatched return of results and investment

• Rapid protein ID testing in 15 min

[patent pending]

• High speed Fc/2 sub domain glycan

profiling for clone selection

• Oligo and peptide synthesis QC

• Free glycan profiling

• Incoming goods QC

Protein assays with 1-2% CV

• LC-free MALDI-TOF immunoassays for

highest assay speed and accuracy [1,2]

Industry leading in Top- Down protein sequencing

• N- and C-terminal sequence readout of 80+

residues at virtually no consumable costs!

• Gold standard for top-down protein

sequencing with terminal clipping variant

identification

• Automated sequence confirmation and

curation [3,4]

• Automated, rapid protein QC reports from

BioPharma Compass software

• Patented T3-Sequencing for direct N- or

C-terminal characterization [5]

• Characterization of PEGylated proteins

Conquer peptide and protein samples too difficult for ESI

• Too hydrophobic or complex proteins

• Non-tryptic peptides, clipping variants

• Large transmembrane or hinge peptides

LC-MALDI – the solution for your difficult challenges

• Automated disulfide crosslink and scrambling

analysis [6,7]

• Top-Down protein sequencing for extensive

IgG sequence confirmation

• Automated assessment of N/O-glycosylation

sites and glycan composition from

glycoprotein digests using GlycoQuest

searches in BioPharma Compass

High Definition MALDI Imaging

• From sample preparation to the molecular

histology workflow: advanced data treatment

and automated tissue classification

employing the best MALDI imaging software

solution: SCiLS Lab

• 2 kHz FlatTop smartbeam-II™ laser for

non-destructive tissue analysis permitting

seamless correlation with high resolution

histopathology from the same tissue slice

[8,9]

Fast and direct results for efficient researchClone selection within 30 minutes from sample collection to result

LC-free MALDI analysis supported in BioPharma Compass:

• Clone selection workflows based on glycan profiles or LC, HC molecular weights

• Sequence validation (intact protein mass, modifications)

• Rapid identity testing for fill and finish operation

• Released glycan profiling (e.g., after 2-AB labelling)

Fc/2 glycan profile analysis of the NIST mAb after IdeS digestion - prepared with 2,5-DAHP matrix and analyzed using the enhanced ultrafleXtreme with 10 bit digitizer. The glycan profile was automatically compared to a reference using the similarity scoring algorithm in BioPharma Compass; the green marked attributes indicate the similarity with the reference profile for clone selection.

Method Attribut Narrow Wide Sample Unit

Target Profile Matching ≥ 0.900 < 0.800 0.984 Similarity [cosine score]

Mass Accuracy [Da] < 3.00 ≥ 8.00 2.17 Da

Oligo QC at unparalleled pace with MALDI MALDI speed provides for the QC of several 1000 samples per day

LC-free MALDI analysis supported in BioPharma Compass:

• Oligonucleotide QC based on molecular weight, sample purity and spectrum profile

• Unexpected species such as early termination variants affect the quality scores

• Spectra acquisition in a few seconds provides for high throughput analysis

• MALDI is well suited for oligos up to 30mers

• BioPharma Compass generates results from ESI and MALDI

Oligonucleotide profile analysis of up to 384 samples from a single AnchorChip MALDI plate in less than 10 min. The MALDI TOF QC analysis automatically checks for molecular weight, purity and relative quantification of typical measurement artefacts such as base losses or adducts.

The ultrafleXtreme meets the urgent needs in biopharmaceutical characterization and can be used for top-down as well as bottom-up approaches. The ultrafleXtreme is the perfect instrument for MALDI experiments - including LC-MALDI. By combining the ultrafleXtreme with the new spotOn fraction collector, simple, seamless LC-MALDI analysis is possible for the first time [10].

The unique ionization characteristic of MALDI opens an exciting dimension to protein characterization.

• Disulfide bond analysis: scrambling, IgG2 isoform determination• Top-down protein sequencing: N/C-terminus verification, clipping analysis• Glycopeptide ID: localization, glycan composition

Advanced Protein Characterizationwith LC-MALDI

Top-down sequencing• MALDI provides the most efficient and

highest performing top-down sequencing

(TDS) method

• In-source-decay allows the direct readout of

up to 100 amino acids from each terminus

providing a sequence coverage of typically

>75%, in less than 5 min.

• N- and C-terminal modifications and

truncations are detected and identified

automatically

Bottom-up • High quality MS/MS spectra enable the ID of

difficult and non-tryptic peptides

• Unique fragmentation pattern from

glycopeptides

• Unique identification of disulfide bond

positions by partial reduction during the

MALDI process leads to identification of

disulfide bonds

• Fast and simple data evaluation including

visual inspection tools

Top-down Bottom-up

spotOn delivers ready to go MALDI sample plates including calibrants and matrices for seamless LC-MALDI analysis

The unique ionization characteristics of MALDI opens an excitingdimension to protein characterization

The spotOn enables automatic and convenient LC-MALDI workflows

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Top-Down Sequencing (TDS)TDS Analysis of Adalimumab

MALDI-TDS is a simple, straightforward sequencing approach providing results at high speed at low cost. The high-quality ISD spectra from the ultrafleXtreme deliver long amino acid sequence readouts with high confidence.

Fraction collection and sample preparation are automatically performed on the new spotOn significantly streamlining LC-MALDI-TDS. TDS results can now be routinely collected for LC separated IdeS digested mAb 25 kDa subunits LC, Fd and Fc/2.

TDS of mAb subunits provides:

• N/C terminal status confirmation

• Confirmation of 2-3 CDRs per variable region

• Modification determination

• Determination of protein clipping variants

• Detection and localization of sequence

aberrations

The LC-MALDI survey

view shows the LC,

Fd and Fc/2 subunits

of Adalimumab, clearly

separated for Top-Down

Sequencing. TDS spectra

are acquired downstream

from each subunit directly

from the same spots on

the MALDI sample plate.

TDS-spectrum analysis of the adalimumab Fc/2 confirms C-terminal lysine clipping and G0F glycan modification. BioPharma Compass automatically matches spectrum and selected sequences and creates an analysis report.

Protein Modification N-term C-term Sequence SVP [%] SC [%] N-term Matching

C-term Matching

Adalimumab fc plus G0F:Adalimumab fc plus G0F fc-G0F:61 yes yes no 67.1 54.8 70 (1-70) 34 (177-210)

Adalimumab fc: Adalimumab fc yes yes no 63.8 52.4 62 (1-62) 34 (177-210)

Adalimumab fc plus K:Adalimumab fc incomplete K clipping

yes no no 43.6 35.1 62 (1-62) 1 (203-203)

Adalimumab LC: Adalimumab LC no no no 10.7. 10.3 1 (9-9) 1 (191-191)

Adalimumab Fd: Adalimumab Fd no no no 9.6 9.2 1 (9-9) 1 (219-219)

Disulfide Bond Analysis

Automated analysis of antibody disulfide bond scrambling

The outstanding sensitivity and dynamic range of the enhanced ultrafleXtreme is the pre-requisite for the detection

of low level scrambling of disulfide bonds. Bruker´s DisulfideDetect software provides an automated disulfide bonds

analysis (DSB) in antibodies with an intuitive visual interface for the analysis of native and non-native disulfide bonds

from a single non-reduced proteolytic digest [11,12].

Detected Disulfide bonds are visualized and scrambled bonds are relatively quantified to the expected bonds in

DisulfideDetect. A) shows the native IgG1 disulfide structure, B) the same sample after heat treatment. The degree

of scrambling of each disulfide bond is indicated by the size of the circles.

Principle of MALDI DSB analysis • Reduction of the disulfide bond occurs in the

MALDI source

• Reduced peptides are identified by MS/MS and

automatically matched using DisulfideDetect

• MS/MS spectra of the corresponding dipeptides as

the one below are used to validate the match

Benefits of LC-MALDI DSB Analysis• Significant time saving through automation

• Digest of non-reduced protein only required

• High specificity resulting from MS/MS analysis

• Semi-quantitative scrambling analysis• No prior assumptions about DSBs required

Get the full picture

The ultrafleXtreme MALDI-TOF/TOF spectra of N-glyco-

peptides reveal clear information of both, the peptide

and the glycan structure.

GlycoQuest uses a MALDI-MS/MS specific fragmen-

tation signature to assign the aglycone and the glycan

masses for further identification via database searches.

Core- and non-core fucosylated glycans can be differen-

tiated due to their specific fragmentation patterns.

Specific signatures also enable the classification and

filtering of O-linked glycopeptides

LC-MALDI-TOF/TOF analysis of a non reduced

mAb digest allows to determine multiple attributes

like disulfide bonds and glycopeptides from a single

LC-MALDI-MS/MS analysis.

Glycopeptide Analysis

LC-MALDI analysis of NIST mAb tryptic digest under non-reducing conditions shows at a glance the N-linked glycopeptide fraction on the MS level (encircled). The same dataset was used for automated disulfide bond analysis .

Automated N-linked core-fucosylated glycopeptide detection of NIST mAb fc glycan G1F by ProteinScape 4.0/ BioPharma Compass 3.0.Signature ion pattern provides molecular weights for both the peptide and the glycan from a single glycopeptide TOF/ TOF spectrum. Structures from both moieties are assigned in the TOF/TOF spectrum.

LC-MALDI workflows

• ProteinScape / BioPharma Compass

incl. GlycoQuest

• DisulfideDetect

Bruker's MALDI-TOF Solutions

Simple, Straightforward, Specific

Read more

1. Popp R, Basik M, Spatz A, Batist G, Zahedi RP, Borchers CH. How iMALDI can improve clinical diagnostics. Analyst 2018,143,2197- 2203.

2. Popp R, Li H, Borchers CH. Immuno-MALDI (iMALDI) mass spectrometry for the analysis of proteins in signaling pathways. Expert Review of Proteomics, 2018 15(9), 701-708.

3. Ayoub D, Jabs W, Resemann A, Evers W, Evans C, Main L, Baessmann C, Wagner-Rousset E, Suckau D, Beck A. Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. MAbs. 2013 Sep-Oct;5(5):699-710.

4. Resemann A, Jabs W, Wiechmann A, Wagner E, Colas O, Evers W, Belau E, Vorwerg L, Evans C, Beck A, Suckau D. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing. MAbs. 2016;8(2):318-30.

5. Suckau D, Resemann A. T³-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrome- try. Anal Chem. 2003;75(21):5817-24.

6. Resemann A, Wunderlich D, Rothbauer U, Warscheid B, Leonhardt H, Fuchser J, Kuhlmann K, Suckau D. Top-down de novo protein sequencing of a 13.6 kDa camelid single heavy chain antibody by matrix-assisted laser desorption ionization-time-of-flight/time-of- flight mass spectrometry. Anal Chem. 2010;82(8):3283-92.

7. Anderson NL, Razavi M, Pearson TW, Kruppa G, Paape R, Suckau D. Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry. J Proteome Res. 2012;11(3):1868-78.

8. https://www.bruker.com/applications/life-sciences/maldi-imaging.html

9. Enomoto H. MALDI-TOF Mass Spectrometry Imaging (MSI) for Distribution Analysis of Anthocyanins in Strawberry Fruits https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/Literature/ApplicationNotes/1867368_MT-120_Analysis_of_Anthocyanins_in_Strawberrys_ebook.pdf

10. spotOn_ebook.pdf. https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/Literature/litera- ture/Brochures/1858938_spotOn_ebook.pdf.

11. Wiesner J, Resemann A, Evans C, Suckau D, Jabs W. Advanced mass spectrometry workflows for analyzing disulfide bonds in biolo- gics. Expert Rev Proteomics. 2015 Apr;12(2):115-23.

12. Resemann A, Liu-Shin L, Tremintin G, Malhotra A, Fung A, Wang F, Ratnaswamy G, Suckau D. Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination. MAbs. 2018;10(8):1200-1213.

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Bruker Daltonik GmbH

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

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

Bruker Scientific LLC

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

For Research Use Only. Not for Use in Clinical Diagnostic Procedures.