An Academic Perspective on the Future of Mass Spectrometry Applications

in the Biotech Industry

Albert J.R. Heck Utrecht University The Netherlandsa.j.r.heck@uu.nlwww.hecklab.com

Introduction to the Heck-lab (www.hecklab.com, now also on twitter)Integrative analysis of glycoproteins by high-resolution native MS and middle-down glycoproteomicsNative high-resolution mass spectrometry for quality assessment of engineered glycoproteins

Analysis of Plasma GlycoproteinsComplement activation studied by mass spectrometry

Biomolecular Mass Spectrometry & ProteomicsUtrecht Institute for

Pharmaceutical Sciences

50-60 people, 22 nationalitiesNearly all continents

Prof. Albert J. R. HeckDr. Maarten Altelaar (Method, Signaling, Cancer Proteomics)Dr Simone Lemeer (Drug Resistance, Cancer Proteomics)Dr. Bas van Breukelen (Computational Proteomics, IT)Dr. Celia Berkers (Metabolomics, Immunology)Dr. Richard Scheltema (Structural Proteomics, Immunology)Dr. Wei Wu (Tumor environment, HLA presentation)Dr. Joost Snijder (Structural Virology, cryoEM and MS)

- 20 mass spectrometers- Computer and IT infrastructure- Cell laboratory + Wet labs- 18 PhDs/15 Post-docs

People – Research - Organization

Integrated approach using combination of analytical chemistry, molecular biology/biochemistry and bioinformatics

Proteomics technology development• New mass analyzers, enrichment techniques, sample preparations• Quantitative proteomics in vivo and in vitro• Quantitative analysis of post-translational modifications• Development of open source bioinformatics tools

Research focus in proteomics applications• Proteome Biology (Cellular signaling, differentiation, stem cells, disease)• Chemical Proteomics; drug interactomes. • Immunology

Metabolomics• Targeting Metabolomic Drug Resistance• Immunometabolomics

Structural Biology• Folding and Transcription Protein Complexes• Structural Biology and Biophysics of Viruses &Bacteriophages• Method Development

Heck-lab: Biomolecular Mass Spectrometry & Proteomics

n Excel in the development of mass spectrometry based enabling technologies for the structural and functional analysis and characterization of proteins and proteomes

n 50/50 in the Departments of Chemistry and Pharmaceutical Sciencesn Bijvoet Institute for Biomolecular Researchn Utrecht Institute for Pharmaceutical Sciences

n Netherlands Proteomics Centre (NGI)n Proteins@Work, X-Omics (Roadmap NWO)n Cancer Genomics.nl (Gravity NWO)n Institute for Chemical Immunology (Gravity NWO)n Prime-XS (FP7), Epic-XS (H2020)

n Thermo, Roche, Pfizer, DSM, Genmab, Crucell, Bruker, ProteinMetrics etc.

Output 40 publications per year with about 30% with other UU/ULS groups

Heck-lab: Mission and embedding

Separation Techniques

Glycan profiling

glycan glycopeptide intact protein

• Chromatographic-based techniques (SCX, HILIC) • Gel electrophoresis (SDS-PAGE, 2D gel) • Capillary electrophoresis

• Mass Spectrometry (MALDI/ESI) • 1D/2D NMR• Glycan differentiation assays/microarrays

CE-MS

MS under denatured conditions

A mass spectrometry-based view into diverse aspects of glycobiology

• Non-denaturing, higher m/z • Less charge states, equally sensitive

Native Mass Spectrometry

1000 2000 3000 4000 5000 6000 7000 8000 9000m/z

6420 6440 6460m/z

23+23+

24+

46+

45+FWHM 46+ : 0.70Resolution : 4600Mass: 148025.83 ± 1.53

FWHM 23+ : 1.25Resolution : 5100Mass: 148025.22 ± 0.56

50/50 water/acetonitrile, 1% f. acid

Aqueous ammonium acetate, pH = 7

Heck Nature Methods 2008

Layout of Exactive Plus

• m/z 400-20,000 in Tune software • Ions trapped in HCD cell• Manual control of gas pressure in the HCD cell• Manual tuning of ion optics

Sprayer

S-lens

Bent flatapole Transport octapole

Injection flatapole

C-TrapHCD multipole

Orbitrap analyzer

Ion gate

Orbitrap Exactive JUMBOmodified for optimal transmission of high m/z ions

Rose et al. Nature Methods 2012Rosati et al. Angew Chemie 2012Rosati et al. mAbs 2013Rosati et al. Nature Protocols 2014

Efficient desolvation and Orbitrap detection allow extremely high mass resolution

m/z

Reso

lutio

n

Exactive Plus instrument resolution at high m/z

0"

5000"

10000"

15000"

20000"

25000"

30000"

35000"

40000"

45000"

50000"

0" 5000" 10000" 15000" 20000"

Theore&cal*

Orbitrap*

Q1ToF*

Assuming R = 150,000 @ m/z = 200

New Detectors for Native MS – UHMR 2017

Quadrupole for isolation of ions up to 40,000 m/z

In-source trapping for desolvation and pseudo MS3 experiments

Improved transport of high m/z ions from the C-trap to the Orbitrap

Extended HCD energies for improved desolvationand tandem MS

Improved focusing of high mass ions throughout the instrument

Nature Methods 14 (2017) 283–286

Used by permission from Thermo Fisher Scientific

Beyond shot-gun: Native and Denaturing Top-Down Proteomics

Intact protein complexes

Intact proteins

T: FTMS + p NSI sid=200.00 Full ms [400.00-12000.00]

5400 5600 5800 6000 6200 6400m/z

5729.66 5958.87

6207.23

5517.37

6476.98

T: FTMS + p NSI sid=200.00 Full ms [400.00-12000.00]

5400 5600 5800 6000 6200 6400m/z

5729.66 5958.87

6207.23

5517.37

6476.982000 3000 4000 5000 6000 7000 8000m/z

5958.95R=1973

5729.69R=1936

6207.30R=1963

5517.40R=1916

6477.17R=2005

5320.48R=1863

6771.96R=1861

Glycosylated antibodies on Orbitrap Exactive Plus 25+

Only few charge statesNo background noiseVery sensitive, attomoles

Single mutations on IgG4 lead to very distinct glycoprofiles

Glycosylation profiles at the intact protein level of four IgG4 mutants Rosati et al. Nature Protocols 2014

What extra brings glycoprofiling of mAbs by native MS

Native MS of Bevacizumab

Presence of co-occurring side products

• Full body with 2 glycan chains• Full body with single glycan chains• Light chain loss• Etc.

BevacizumabAvastin, Roche

Native Mass Spectrometry

Native mass spectrometry services providingthe expertise, equipment and labor

for the analysis of intact protein and protein complexes

Interested? Come and see my poster!

Dominique Hagemans, Arjan Barendregt

Sequence Mass: 42750.1 (385 aa)Sequence: P01012 uniprotAverage Mass: ~45 kDa

Initiator methionine is removed.

A Proteotypic Protein: chicken ovalbumin

Unprocessed Deglycosylated

Dephosphorylated Deglycosylated &Dephosphorylated

A Proteotypic Protein: chicken ovalbumin

Signals are focused in two charge-states. Ovalbumin can be treated by phosphatase (CIP) and/or deglycosylase

Incomplete glycosylation: Endo F1 Specificities

Zoom in on [M+13H]13+ m/z 3278-3562

Starting mass : 42995.22

Unprocessed Deglycosylated

Dephosphorylated Deglycosylated &Dephosphorylated

A Proteotypic Protein: chicken ovalbumin

Unprocessed

Dephosphorylated

Full Glycoform profiling: chicken ovalbumin

62 peaks deconvoluted, 45 match the search30 have been annotated

68 peaks deconvoluted59 have been profiled including 1 N-acetylation cleavage;2 single phosphorylation sites45 glycoforms.

Without * previously assigned, with * are novel never been identified structures (~20/45)

Qualitative and semi quantitative glycan profiling

Yang et al. Anal Chem 2013

Aids in glycan assignmentsReveals quantitative validity of the measurements

Chopping up intact mAbs monitored by high-resolution native MS

Rosati et al. Nature Protocols 2014

Overall glycoprotein profile by native MSSite-specific profile of PTMs per site

Integrating High-Resolution Native Mass Spectrometry and Middle-Down Proteomics for the Analysis of Glycoproteins

“The whole is greater than the sum of its parts” Aristotle

Over 230 peaks could be base-line resolved, and the overall PTM composition could be assigned with satisfying mass accuracyObserve minor modifications of sialic acid by O-acetylation and –CH3 to –CH2OH replacement

High-Resolution Native Mass Spectrum of human Erythropoietin

EPO backbone = 18,235.99 Da, Measured Mw by native MS between 26,000 to 33,000 Da

Loss of 13 sialic acids, heterogeneity stems largely from sialic acid

High-Resolution Native Mass Spectrum of human ErythropoietinSialidase treated

Identified and relatively quantified 1) 10 glycoforms on N24, 2) 9 glycoforms on N38, 3) 8 glycoforms on N83 and 4) 2 glycoforms on S126

Each N-glycosylation site on rhEPO is modified uniquely in terms of both the numbers and relative abundances of differentially modified glycoforms

Middle-down analysis of human Erythropoietin

Glycopeptides may easily lose their labile sialic acid moiety during sample preparation and ionization

Is the whole greater than the parts

PTM heterogeneity in rhEPO products is largely originating from the variability in the extent and occupancy of sialylation on the various glycan trees occurring in rhEPO

An Erythropoietin Biosimilarity Score

Gene-editing tools allow us to custom-engineer, but what about the read-out?

Glyco-engineering : custom-engineered EPO

Gene-editing tools allow us to custom-engineer, but what about the read-out?

Glyco-engineering : custom-engineered EPOEPO code name Gene KO EffectEPO WT / Wild typeEPO 01 Mgat1 KO paucimannose glycansEPO 02 Mgat3/4a/4b/5 Cosmc KO Biantennary glycans and truncated O-glycansEPO 03 ST3Gal4/6 KO Loss of N-glycan sialylationEPO 04 Mgat2 KO Loss of β2-Branch

EPO 05 Mgat4a/4b/5 St3Gal4/6 KO + ST6Gal2 KI Homogenous biantennary α2,6-NeuAc sialylated glycansEPO 06 B3gnt2/mgat4a/4b/5 KO Biantennary glycans with eliminated polyLacNAcEPO 07 Mgat4a/4b/5 Biantennary sialylated glycans

EPO 08 Mgat4a/4b/5 ST3Gal4/6/B3gnT2 Biantennary, no Sialylation on N-glycans, no polyLacNAcEPO 09 B4GalT3 KO No effectEPO 10 B3GnT1 KO No effectEPO 11 B3Gnt2 KO No polyLacNAcEPO 12 B4GalT4 KO No effectEPO 13 Mgat3/4a/5 + Cosmc KO Similar to mgat5 KO but with truncated O-glycansEPO 14 Mgat 5 KO Eliminates β6-branch of N-glycansEPO 15 Mgat4a/4b KO Eliminates β4-branch of N-glycansEPO 16 Mgat4a/4b/5/ST3Gal4/6 cosmc KO UNKNOWN(sample mislabeled)

Gene-editing tools allow us to custom-engineer, but what about the read-out?

Custom-engineered EPO: Mgat5 versus Mgat 4a/4b example

Mga

t5

Mga

t4a/

4b

Custom-engineered EPO: Mgat5 versus Mgat 4a/4b example

Each glyco-enzyme deletion has a distinct effect on the glycosylation

β-6 branch is a preferred acceptor site for LacNAc elongation

Mgat5 vs Mgat 4a/4b

An Erythropoietin Biosimilarity Score: EPO barcodes

Engineered EPOs cluster according to their branching characteristics

TomislavCaval

FanLiu

Zhang Yang

HenrikClausen

VojtechFranc

Yang Yang

Acknowledgement

Analysis of Plasma Glycoproteins

Albert J.R. Heck Utrecht University The Netherlandsa.j.r.heck@uu.nlwww.hecklab.com

What is a protein ?

Overall glycoprotein profile by native MSSite-specific profile of PTMs per site

Integrating High-Resolution Native Mass Spectrometry and Middle-Down Proteomics for the Analysis of Glycoproteins

“The whole is greater than the sum of its parts” Aristotle

Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity.Yang Y, et al. Nat Commun. 7 (2016) 13397.

Human Fetuin

fetuin is synonymous with α2-HS-glycoprotein

Structure of mammalian plasma fetuin-B and its mechanism of selective metallopeptidase inhibition. Cuppari A. et al.IUCrJ. 6 (2019) 317-330. doi: 10.1107/S2052252519001568.

Proteoform profiles of fetuin from different sources

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human FetuinLin YH et al., J Proteome Res. 2018 Aug 3;17(8):2861-2869

Proteoform profiles of fetuin from different sourcesGlycopeptide analysis

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human FetuinLin YH et al., J Proteome Res. 2018 Aug 3;17(8):2861-2869

Proteoform profiles of fetuin from different sourcesDifferences in phosphorylation, fucosylation and backbone-processing

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human FetuinLin YH et al., J Proteome Res. 2018 Aug 3;17(8):2861-2869

Proteome plasma profiles of single donors

Utrecht Approach:

One gene at the time

Zooming in on plasma glycoproteins

Many major proteins in plasma are glycoproteins, diagnostic for the Glyco-profile of individuals

fetuin is synonymous with α2-HS-glycoprotein

Separation and purification of plasma glycoproteins

Use combination of cation/anion exchange chromatography, 100 uL of plasma

fetuin is synonymous with α2-HS-glycoprotein

Proteoform profiles of human fetuin isolated from a single donor

Proteoform profiles of fetuin from individual donors

Proteoform profiles of fetuin from different genotypes

Differences in Ser/Thr O-glycosylation

m/z

AHSG*1AHSG*2

Extensively O-glycosylated

Nearly not O-glycosylated

Proteoform profiles of fetuin from different genotypes

AHSG*1AHSG*2

Differences in Ser/Thr O-glycosylation

A. Unsupervised clustering of proteoform profiles 10 healthy individualsB. Clustering on specific signature peaks defines the genotypes Donor F5 (AHSG*1) and M5 (AHSG*2) are outliers caused by excessive fetuin phosphorylation C. Clustering of fetuin proteoform profiles derived from 10 healthy and 10 septic patients

Orange: AHSG*2Blue: AHSG*1Green: AHSG1/2Purple: Septic Patient

Clustering of personalized proteoform profiles

Fetuin fucosylation is biomarker for sepsis

Significantly increased fucosylation at site N176, but less prominent in younger people

AcknowledgementsSara RosatiGuanbo WangMaurits den BoerVojtech Franc

Rob de JongEwald van den BremerJanine Schuurman

Alexander Makarov

Deniz UgurlarPiet Gros