unraveling the structural heterogeneity of recombinant ... · – andy hanneman (charles river) ......

Jason C. Rouse Pfizer, Inc., Biotherapeutics Pharmaceutical Sciences, Andover, MA September 10, 2018

Unraveling the Structural Heterogeneity of Recombinant Protein Therapeutics with High-Resolution Mass Spectrometry: A Personal Journey

MASS SPEC 2018: 15th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry, San Francisco, CA

Theme: Answer where we have been and where we are going?

My Journey in MS Started at MSU The triple quadrupole turns 40...

C&E News: March 5, 2018 Vol 96:10 | pp. 15-18

Chris Enke Rick Yost

Circa 1978

Prof. John Allison Dr. Karen Wahl Dr. Jeff Gilbert

My Ph.D. Thesis: 1. Studied ion formation mechanisms in

fast-atom bombardment (FAB) 2. Studied mechanistic & thermochemical

aspects of peptide fragmentation 3. Fragmented metal-cationized small

molecules/peptides via FAB-CAD MS/MS

Genetics Institute Post-Doc (1993-1995) • My Research Projects

– Optimized continuous-flow FAB on JEOL HX-110/HX-110 4-sector mass spectrometer

– Compared MALDI-PSD to high and low energy CAD on JEOL HX-110/HX-110 4-sector mass spec

– Developed MALDI cleanup methods for analysis of released N-linked glycans

– Elucidated N-linked glycan isomers by MALDI, PSD and glycosidases

• Structural Biochemistry - MS group – Hubie Scoble, Director (Sanofi) – Steve Martin (Waters) – James Vath (Zafgen) – Wen Yu (MedImmune) – Mike Huberty – Stephan Koza (Waters) – Bernice Yeung (Postdoc Shire) – Aston Liu (GSK) – Paul Russo (PostdocGeorge Mason U)

• Carry-out project-based structural characterization and comparability – Determine primary structure and posttranslational/chemical modifications – Support forced degradation and structure-function studies

• Support process and product investigations as needed • Maintain all mass spectrometers • Continuous improvement: investigate new instruments, methods & technologies La

b M

issi

on

& Protein Mass Spec Groups – Mellisa Zolodz – Justin Sperry (PFE ARD PPL) – Paul Brown (PFE ARD MS) – Olga Friese (PFE ARD MS) – Dan Haq (Merck-Serono) – Uma Raut (Shire) – Kathleen Cornelius (PFE ARD MS) – Jacky Smith (PFE ARD MS) – Sasidhar Nirudodhi (Post-doc, PFE Vaccines) – Mellisa Ly (PFE ARD MS) – Michelle English (Protein Metrics) – Elaine Stephens (Visiting, PFE ARD MS) – Ying Zhang (PFE ARD MS) – Andrew Dawdy (PFE ARD MS) – Thomas Powers (PFE ARD MS) – Kristin Boggio (PFE ARD MS) – Roger Theberge (Visiting, PFE ARD MS) – Leah Wang (PFE ARD MS) – Brian Gau (PFE ARD MS) – Peilu Liu (PFE ARD MS)

• Wyeth (2000’s) – Jennifer Nemeth (Janssen) – Lisa Marzilli (PFE ARD MS) – Lisa Sapp (Agilent) – Keith Johnson (PFE ARD PPL) – Tim Keefe (Acceleron) – Carrie Davis (Microbac Labs) – Joe McClellan (PFE Biosims) – Promod Mehndiratta (Celgene) – Heather DeGruttola (PFE ARD MS) – Kelly Newton Toler (Post-doc) – Chris Taylor (Cambridge Iso) – Alexei Colin (Summer intern) – Andy Hanneman (Blue Stream) – Matt Thompson (PFE ARD MS) – Andrew Saati (PFE ARD MS)

• Pfizer (2010’s) – James Carroll (PFE ARD PPL) – Rob Dufield (PFE ARD QC)

& Protein Mass Spec Groups – Mellisa Zolodz – Justin Sperry (PFE ARD PPL) – Paul Brown (PFE ARD MS) – Olga Friese (PFE ARD MS) – Dan Haq (Merck-Serono) – Uma Raut (Shire) – Kathleen Cornelius (PFE ARD MS) – Jacky Smith (PFE ARD MS) – Sasidhar Nirudodhi (Post-doc, PFE Vaccines) – Mellisa Ly (PFE ARD MS) – Michelle English (Protein Metrics) – Elaine Stephens (Visiting, PFE ARD MS) – Ying Zhang (PFE ARD MS) – Andrew Dawdy (PFE ARD MS) – Thomas Powers (PFE ARD MS) – Kristin Boggio (PFE ARD MS) – Roger Theberge (Visiting, PFE ARD MS) – Leah Wang (PFE ARD MS) – Brian Gau (PFE ARD MS) – Peilu Liu (PFE ARD MS)

• Wyeth (2000’s) – Jennifer Nemeth (Janssen) – Lisa Marzilli (PFE ARD MS) – Lisa Sapp (Agilent) – Keith Johnson (PFE ARD PPL) – Tim Keefe (Acceleron) – Carrie Davis (Microbac Labs) – Joe McClellan (PFE Biosims) – Promod Mehndiratta (Celgene) – Heather DeGruttola (PFE ARD MS) – Kelly Newton Toler (Post-doc) – Chris Taylor (Cambridge Iso) – Alexei Colin (Summer intern) – Andy Hanneman (Charles River) – Matt Thompson (PFE ARD MS) – Andrew Saati (PFE ARD MS)

• Pfizer (2010’s) – James Carroll (PFE ARD PPL) – Rob Dufield (PFE ARD QC)

MS group: 22 colleagues

15th Anniversary of CASSS Mass Spec Conference !!

2006 Software

2013 HOS Method Comparison

2016 MAM, Hotspot Analysis & in vivo Biotransformation

2017 Viral Capsid Characterization

and CDMS

2018 MS Software 2016 CE-MS

Technology Update

2013 MS in QC; Introduction to MAM

2012 Vaccines

2013 Host cell protein proteomic analysis

2013 Biosimilars 2016 Top-down MS progress

2016 De Novo Sequencing

2018 ADC characterization

2015 Trisulfides

2014 Regulatory

2014-2018


2012 Sequence Variants & HCPs

2011 Biosimilars

2011 ADCs

2011 UHR MS

2010 N-linked glycans, SVs &

trisulfides

2008 Biosimilars and N-glycans

2008 Pharmacokinetics

2007 High Throughput MS & Process Characterization

2006 Forced Degradation by MS

2007 Protein Stability & Fragmentation

2007 Comparability

2005 QC & PAT

2005 Comparability

2005 Product Characterization

2004-2008

2005 N-glycans

2009-2013

2004 MS in QC

2004 MS Apps: Top-down vs bottom up

Greg Adams (Diosynth Biotech) Scott Buckel (Amgen) Steven Cohen (Waters) John Dougherty (Eli Lilly & Co) Daotian Fu (Genzyme) William Hancock (Barnett Inst.) Victor Ling (Genentech) Rohin Mhatre (Biogen Idec) Anthony Mire-Sluis (Amgen) Jason Rouse (Wyeth BioPharma)

Michael Boyne (COUR Pharma) Steven Cohen (SAC Analytical) Terry Cyr (Health Canada) Ingo Lindner (Roche Diag.) Anders Lund (Synlogic) Yelena Lyubarskaya (Sanofi) Frances Namuswe (CDER, FDA) David Passmore (RubrYc Therapeutics) Richard Rogers (Just Biotherapeutics) Sarah Rogstad (CDER, FDA) Jason Rouse (Pfizer) Arjen Scholten (Janssen) John Valliere-Douglass (Seattle Gene.) Christopher Yu (Genentech)

Greg Adams (FUJIFILM Diosynth) Alain Balland (Amgen) James Carroll (Pfizer) Steven Cohen (Waters) Victor Ling (Genentech) Anders Lund (Genzyme) Yelena Lyubarskaya (Biogen Idec) Jun Park (CDER, FDA) David Passmore (BMS) Dietmar Reusch (Roche Diag.) Jason Rouse (Pfizer) Hansjörg Toll (Sandoz)

15th Anniversary of CASSS Mass Spec Conference !!

2006 Software


2016 MAM, Hotspot Analysis & in vivo Biotransformation

2017 Viral Capsid Characterization

and CDMS

2018 MS Software 2016 CE-MS

Technology Update

2013 MS in QC; Introduction to MAM

2012 Vaccines

2013 Host cell protein proteomic analysis

2013 Biosimilars 2016 Top-down MS progress

2016 De Novo Sequencing

2018 ADC characterization

2015 Trisulfides

2014 Regulatory

2014-2018


2012 Sequence Variants & HCPs

2011 Biosimilars

2011 ADCs

2011 UHR MS

2010 N-linked glycans, SVs &

trisulfides

2008 Biosimilars and N-glycans

2008 Pharmacokinetics

2007 High Throughput MS & Process Characterization

2006 Forced Degradation by MS

2007 Protein Stability & Fragmentation

2007 Comparability

2005 QC & PAT

2005 Comparability

2005 Product Characterization

2004-2008

2005 N-glycans

2009-2013

2004 MS in QC

2004 MS Apps: Top-down vs bottom up

The Rise of Mass Spec in Biotech • 1990’s

– First generation commercial MALDI-TOF and ESI instruments • Intact glycoprotein analysis and online LC/MS – peptide mapping emerged

• Lower resolution & mass accuracy compared to 1980’s MS techniques

• Huge leap in capability & sensitivity, advancing protein/product characterization

• 2000’s – High-resolution ESI-quadrupole time-of-flight & Orbitrap MS emerged

• Afforded accurate mass determinations of proteins/peptides with high sensitivity

• Brought about modern product characterization & comparability approaches

• 2010’s – Era of ultrahigh-resolution MS for product characterization/comparability

• Unprecedented increases in sensitivity, speed, resolution & mass accuracy

• Bioprocess support for sequence variant & host cell protein analyses began

35000 45000

23970 23990

51000 51600

Key MS Platform Advances for rProtein Characterization

Date Vendor Instrument Configuration Advance

1992 Bruker Daltonics Reflex I MALDI-TOF Low pmol sensitivity for peptides / proteins / glycans

1997 Thermo Finnigan LCQ ESI-QIT Online LC/MS-peptide mapping becomes routine

2000 Waters Q-Tof-2 ESI-QTOF Offline HR/AM nanoESI MS and online HR/AM LC-MS for peptides / proteins / glycans

2005 Thermo Scientific LTQ-Orbitrap XL ESI-LIT-orbitrap

HR/AM & superior sensitivity for proteomics era

2009 Bruker Daltonics maXis ESI-QTOF HR/AM & superior sensitivity Subunit monoisotopic masses

2014 Thermo Scientific Exactive+ EMR ESI-orbitrap Extended mass range for native spray SEC-MS/CEX-MS

2014 Thermo Scientific Orbitrap Fusion ESI-orbi-LIT Superior proteomic SV and HCP analysis plus site-specific characterization via HCD, ETD 2017 Thermo Scientific Orbitrap Lumos ESI-orbi-LIT

High-resolution / accurate mass (HR/AM)

Topics

• Genetics Institute post-doctoral research (1990’s) – Elucidation of N-glycans – isomer heterogeneity – Beginnings of on-line N-glycan analysis

• Wyeth (2000’s) – Top-down characterization strategy w/ESI-QTOF MS – Commercial product comparability – PTM heterogeneity

• Pfizer (2010’s) – mAb CDR hotspot analysis – the invisible heterogeneity

Matrix-Assisted Laser Desorption/Ionization(MALDI) TOF MS

Bruker Reflex TOF-MS 337 nm UV Laser

28.5 kV 23.5 kV

0.0 V 0.0 V

HO

O

C OH

3,5-methoxy-4-hydroxycinnamic acid (6 mg in 500 µL 33% ACN/0.1% TFA)

SA matrix

OCH3 H

H CH3O 1 2

Deposit purified protein sample (1-5 pmol) & let dry

Deposit 1 μL of SA matrix on dried sample

MALDI Carbohydrate Mass Profiling (1994)

Rouse and Vath. Anal. Biochem. 1996, 238, 82.

On-probe cleanup w/ cation-exchange resin

(NH4+ form)

Equilmolar mixture SDHB matrix

R/A rhBMP-2 SDHB matrix

PNGaseF

On-probe cleanup

Profiling EndoH-released N-glycans by HPAEC

High-pH anion exchange chromatography with pulsed electrochemical detection: • Dionex CarboPac PA-100

column • MPA & MPB: 0.1M NaOH • MPB: 0-500 mM NaAcetate • 1 mL/min

Rouse and Strang (1996), ASMS Conference 1998

MALDI-TOF MS Analysis of rFVIII HPAEC Fractions

AMMS-II

Fractions

Dionex BioLC HPAEC-PED


MALDI-PSD-TOF MS of Man7 Branching Isomers

O O

O

0 1

O 4

3 2

5 6-arm

3-arm

Rouse, Strang, Yu, Vath. Anal. Biochem. 1998, 256, 33. Rouse and Strang (1996), ASMS Conference 1998

MALDI-PSD of Minor Co-eluting Species in Fr. 6


N-glycan Composition Possibilities for Fr. 12

6

3

6

3

3


Sequential Glycosidase Digestion of Fr. 12

Dr. Terry D. Butters, Oxford Glycobiology Institute


MALDI-PSD Comparison of Man8 & Fr. 12


O O

O

0 1

O 4

3 2

5 6-arm

3-arm

Elucidation of HPAEC-Separated rFIX N-Glycans via Off-Line ESI, MALDI, PSD and Glycosidases

GlcNAc

Fuc

ManGal

NeuAc

oror oror

or or

+

+ + +

oror oror

or or

+

+ +

+

++

+

+ + ++

+ + ++

+ + +

Anne-Marie Strang, Kate Seaber, Bi Xu, and Jason Rouse, 1995-1996

Rouse, Strang, Yu, Vath. Anal. Biochem. 1998, 256, 33.; Rouse, Strang, Liu, Hardy, Scoble, ASMS Conference 2000

LC/MS Capabilities in Andover MS Lab (late 1990’s)

New Objective PicoTip Interface for HPAEC/MS 4.2 kV 220°C

100 µm I.D. Taper Tip

AMMS-II

H2SO4 (gradient,

10 mL/min) Fraction Collector 984 µL/min

16 µL/min 2-5 mm

gap Thermo Finnigan

LCQ ESI-QIT

MS

Dionex BioLC HPAEC-PED CarboPac PA100

1 ml/min A: 100 mM NaOH B: 0-500 mM NaOAc 100 mM NaOH

Rouse, Strang, Liu, Hardy and Scoble, ASMS Conference 2000; Long Beach, CA

HPAEC/MS of rFIX N-Glycans with New Objective Interface RT: 5.00 - 50.00 SM: 7B

5 10 15 20 25 30 35 40 45 50Time (min)

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive

Abun

danc

e

NL:2.46E5Analog UV 1

RT: 5.00 - 50.00 SM: 7B

5 10 15 20 25 30 35 40 45 50Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rela

tive

Abun

danc

e

NL:7.38E7m/z= 1000.0-2000.0

HPAEC-PED

LCQ RTIC 2NeuAc-BiF Mobs=2368.4 Mcalc=2368.8

A

B C D

4NeuAc-TetraF Mobs=3681.0 Mcalc=3681.3

3NeuAc-L2-TriF Mobs=3754.2 Mcalc=3755.3

3NeuAc-TriF Mobs=3024.6 Mcalc=3025.1

3NeuAc-L1-TriF Mobs=3390.0 Mcalc=3390.2

1NeuAc-TetraF Mobs=2807.6 Mcalc=2808.0 1NeuAc-TriF Mobs=2442.6 Mcalc=2442.9 4NeuAc-TetraF

Mobs=3681.0 Mcalc=3681.3

4NeuAc-L2- TetraF

Mobs=4410.9 Mcalc=4411.6

4NeuAc-L1-TetraF

Mobs=4046.4 Mcalc=4046.4

4NeuAc-L3- TetraF

Mobs=4776.0 Mcalc=4776.7

2NeuAc-TriF Mobs=2733.4 Mcalc=2734.0

2NeuAc-TetraF Mobs=3098.6 Mcalc=3099.1

Rouse, Strang, Liu, Hardy, Scoble, ASMS Conference 2000

Selected Mass Spectra from HPAEC/MS of rFIX N-Glycans

Rouse, Strang, Liu, Hardy, Scoble, ASMS Conference 2000

Criteria for Online MS Characterization of Released N-Glycans

• Develop a LC-MS method to interface with the N-glycan profiling methods with minimal impact on chromatographic separation – Easy to set-up for routine operation – Stable during the course of a run – Robust performance run-to-run – Total ion chromatogram matches fluorescence profile

• Maximize sensitivity – Facilitate analysis of low-level N-glycans to <0.5% – Minimize signal splitting due to formation of multiple glycan ion types – Minimize column shedding effects

• Minimize artifacts – Gas-phase fragmentation

• (m/z 204, 366, 657) – Electrochemical degradation

[M+2H]2+

m/z

m/z

[M+2H]2+ [M+Na]+ [M+2Na]2+

[M-H+2Na]+

[M+H+Na]2+

[M+H]+

HILIC/MS for mAb-released N-glycans: Shang, Saati, Toler, Mo, Li, Matlosz, Lin, Schenk, Ng, Duffy, Porter, Rouse J Pharm Sci. 2014; 103: 1967.

Industry Moved to 2-AB Labeling of Released N-glycans for Characterization & Quantitative Assessment via LC/FLR/MS

Time (minutes)

Fluore

scenc

e (mV

)

0.0

4000.0

8000.0

12000.0

24.00 36.00 48.00 60.00 72.00 84.00

0.00

600.00

1200.00

1800.00

24.00 36.00 48.00 60.00 72.00 84.00

Full View

Enhanced View

1 2 1

2

3

1

2

3

4

5

1

2

3

4, 5 8

31

26 7

Neutral region

Monosialylregion

Disialylregion

Trisialylregion

3

45

6

4

Tetrasialyl region Direct LC/FLR/MS N-linked Glycan Profiling of Recombinant Factor IX (rFIX)

Region Peak Observed Mass (Da) Theoretical Mass (Da) Mass Error (ppm) Proposed Structure Tetrasialyl 1 4896.92 4896.73 39 4A / 3R / 4SA / 1F

2 4531.82 4531.63 42 4A / 2R / 4SA / 1F 3 4208.54 4209.51 230 4A / 1R / 4SA / 1F + O-acetyl 4 4167.02 4166.50 125 4A / 1R / 4SA / 1F 5 4167.02 4166.50 125 4A / 1R / 4SA / 1F 6 3843.38 3843.38 0 4A / 0R / 4SA / 1F + O-acetyl 7 3947.42 3947.42 0 4A / 0R / 4SA / 2F 8 3801.32 3801.37 13 4A / 0R / 4SA / 1F

Adapted from Anumula & Dhume Glycobiology 1998;

8(7), 685.

Himakshi Patel and Matt Thompson, 2008-2009; LC/FLR/MS via Thermo Scientific LTQ in negative ion mode

DOE-optimized sample prep Standard NP-HPLC with MS-compatible mobile phases New NH2-bonded column

Industry Moved to 2-AB Labeling of Released N-glycans for Characterization & Quantitative Assessment via LC/FLR/MS

Time (minutes)

Fluore

scenc

e (mV

)

0.0

4000.0

8000.0

12000.0

24.00 36.00 48.00 60.00 72.00 84.00

0.00

600.00

1200.00

1800.00

24.00 36.00 48.00 60.00 72.00 84.00

Full View

Enhanced View

1 2 1

2

3

1

2

3

4

5

1

2

3

4, 5 8

31

26 7

Neutral region

Monosialylregion

Disialylregion

Trisialylregion

3

45

6

4

Tetrasialyl region Direct LC/FLR/MS N-linked Glycan Profiling of Recombinant Factor IX (rFIX)

DOE-optimized sample prep Standard HPLC with MS-

compatible mobile phases New NH2-bonded column

Adapted from Anumula & Dhume Glycobiology 1998;

8(7), 685.

Himakshi Patel and Matt Thompson, 2008-2009; LC/FLR/MS via Thermo Scientific LTQ in negative ion mode

1000 1200 1400 1600 1800 2000 m/z

Rel

ativ

e Ab

unda

nce

1266.099

1899.652

Peak 8 4A/0R/4SA/1F [M-3H+]3- [M-2H+]2-

-2AB -NeuAc

-2AB -NeuAc Na+

1000 1200 1400 1600 1800 2000 m/z

Rel

ativ

e Ab

unda

nce

1387.999

Peak 5 4A/1R/4SA/1F

[M-3H+]3-

-2AB -NeuAc Na+

ESI-Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF)

Quadrupole Mass Analzyer

Time-of-Flight M

ass Analyzer

Z-Spray ESI Source

Hexapole Ion Bridge

Quadrupole Mass Filter

Hexapole Collision

Cell

Ar Gas

Reflectron

Pusher MCP

Detector

Orthogonal Acceleration Region Cone Extractor

ESI Probe

Q-Tof is well suited for characterizing glycoproteins...

high resolving power (~11,000 in V-mode) high mass accuracy: <30 ppm for peptides & N-glycans (NaI ext. cal. & DXC) <50 ppm for intact proteins wide usable “mass” range (m/z 50-4000) efficient ion transmission nano-, micro-, and macroESI (LC/MS) capable effective MS/MS for peptides, N-glycans, & intact proteins powerful MaxEnt-1 & MaxEnt-3 deconvolution software

Adapted from schematic of Q-Tof-2 (Micromass MS Technologies, Waters Corp.)

Parts-per-million mass accuracy:

[Mr (Obs.) – Mr (Theor.)] x 106

Mr (Theor.)

Top-down Characterization Strategy for rProteins

Reduce complexity

Bulk sample

Direct accurate mass analysis of intact protein with all polypeptide chains and PTMs

Correlate data from all methods

Decrease molecular size: - Reduction/alkylation, limited proteolysis, etc. (i.e., C4 RP-HPLC) Decrease molecular heterogeneity: - Remove N-glycans / sialic acids (glycosidases) (i.e., C4 RP-HPLC) - Fractionate and enrich isoforms (i.e., cation-exchange-HPLC)

Peptide mapping N-glycan profiling

Specific structural details

Data analysis

(i.e., nanoESI-QTOF MS RP-HPLC/QTOF MS SEC-QTOF MS)

(i.e., C18 RP-HPLC/MS) (i.e., HILIC/MS)

Kelleher, Lin, Valaskovic, Aaserud, Fridriksson, McLafferty. J. Am. Chem. Soc., 1999, 121, 806. Kelleher, N. L. “Top-Down Proteomics” Anal. Chem. 2004, 76, 196A.

rFIX Comparability Case Study: Added New DS Manufacturing Site

• Expanded commercial DS manufacturing capacity for rFIX was required

• Comparability program was necessary to ensure product quality, safety, and efficacy as a surrogate for clinical trials – Process performance, DS, DP, and a non-clinical, rat PK study

– Analyze pre-change and post-change samples from beginning, end, and middle of inoculum

• Analytical release testing and heightened characterization testing – Complex acceptance criteria and statistical analysis of release testing data

– Added heightened characterization testing and structural analysis of rFIX

– All samples were run side-by-side and directly compared

• Defined pre-determined acceptance criteria for MS-based structural comparability – MS analysis is not routinely performed as part of product release testing

– Set based on instrument specifications and qualitative isoform content

Structure of rFIX

Activation Peptide

Serine Protease Domain

Gla Domain

EGF-1 EGF-2 Post-translational modifications N- and O-linked glycans 4-carboxyglutamic acid (Gla) β-hydroxy-aspartic acid Sulfation

Post-translational processing COOH-terminus

Zero-charge Mass Spectrum of Intact rFIX

53000 53500 54000 54500 55000 55500 56000 56500 57000 57500mass0

100

%

55615.0 rFIX (LC-AP-HC) 11 disulfide bonds 12 4-carboxyglutamic acids (Gla) 1 Glc-Xyl-Xyl O-glycan 1 Fuc-GlcNAc-Gal-NeuAc O-glycan 2 Tetraantennary N-glycans (4 NeuAc,1 Fuc) = 55614.0

+Gal-GlcNAc

+Gal-GlcNAc

+Gal-GlcNAc

+Gal-GlcNAc + 2 Classical

O-glycans

TetraF / TetraF

TriF / TetraF

TriF / TriF

BiF / TriF

∆ 1 Gla

∆ 2 Gla

∆ 1 β-OH

∆ NeuAc ∆ NeuAc

C4 RP-HPLC / ESI-QTOF MS

rFIX is highly heterogeneous due to expected PTMs

McClellan, Steinmeyer, Gallant, Patel, Jankowski, Porter, and Rouse, CASSS Mass Spec Conference 2005

RP-HPLC separation of the rFIX activation products

Pre-determined acceptance criteria Comparable RP-HPLC chromatograms

of rFIX activation products Agreement of masses for analogous

isoforms in each mass spectrum Measured masses must agree within 50

ppm of the theoretical masses Similar peak distributions for analogous

mass spectra No new species are detected and no

expected species are absent

Rouse, McClellan, Patel, Jankowski, and Porter. (2005) Methods in Molecular Biology, vol. 308: Therapeutic Proteins: Methods & Protocols, 435.

N-glycans + O-glycans

N-glycans (only)

rFIXa

Comparability of FIXa by RP-HPLC/QTOF MS

Rouse, McClellan, Patel, Jankowski, and Porter. (2005) Methods in Molecular Biology, vol. 308: Therapeutic Proteins: Methods and Protocols, 435.

Pre-determined acceptance criteria Comparable RP-HPLC

chromatograms of rFIX activation products

Agreement of masses for analogous isoforms in each mass spectrum (within ≤2 Da)

Measured masses must agree within 50 ppm of theoretical masses

Similar peak distributions for analogous mass spectra

No new species are detected and no expected species are absent

CV = 37 V CE = 10 eV

Comparability of AP by RP-HPLC/QTOF MS Pre-determined acceptance criteria Comparable RP-HPLC

chromatograms of rFIX activation products

Agreement of masses for analogous isoforms in each mass spectrum (within ≤1 Da)

Measured masses must agree within 50 ppm of theoretical masses

Similar peak distributions for analogous mass spectra

No new species are detected and no expected species are absent

Rouse, McClellan, Patel, Jankowski, and Porter. (2005) Methods in Molecular Biology, vol. 308: Therapeutic Proteins: Methods and Protocols, 435.

CV = 20 V CE = 5 eV

Summary of rFIX Comparability Results • RP-HPLC/ESI-QTOF MS provides unique structural endpoints

– Confirms amino acid sequence composition – N- and O-glycosylation heterogeneity – Number of Gla modifications present

• Results show that rFIX DS materials are comparable – Chromatographic profiles are highly similar – No new species were observed and none were absent – Analogous isoforms had mass agreement – All observed masses were within mass error specifications – Only a slight redistribution of isoforms was observed

• Data indicate that there is no significant impact of the manufacturing site change on the structural isoforms of rFIX

• This effort helped to get the new manufacturing facility approved

mAb LC/MS – Subunit Analysis via QTOF MS Light Chain Heavy Chain

Wat

ers

Q-T

of-2

B

ruke

r maX

is

mass 50800 51000 51200 51400 51600

%

0

100 51389.8 51228.5

51081.0 50999.7

51551.4 Man5

G0

G0F G1F

G2F

51000.823 51082.264

51228.430 51390.468

51552.504

50800 51000 51200 51400 51600

23743.610

23730 23740 23750 23760

23743.586

23720 23730 23740

Theoretical mass and isotopes

-13 ppm

+6 ppm -10 ppm

+1 ppm +0.1 ppm -2 ppm

mass 23730 23740 23750 23760

%

0

100 23743.9

Theoretical L chain mass (ave): 23744.2

Porter and Rouse; CASSS HPLC 2010, Boston, MA

Platform Methods for Heightened Characterization

Intact Mass

Subunit Analysis

Peptide Mapping

N-Glycan Profiling

Multi-chain molecular structure;

Product isoforms / conjugate forms

Primary structure and PTMs;

Product isoforms / conjugate forms

Primary structure; site-specific char. of

PTMs, chemical modifications,

linker-payloads…

N-linked glycan structures and quantification

G0F/G0F149268.1

mass147500 148000 148500 149000 149500 150000 150500 151000

%

0

100

1

G0F/G1F149430.6

G0/G0F G1F/G1F2 3

H20 H24

H12

H13

L12 L6

L15

H30

, H10 L9

L10

H3

H23

H32

L14

H26

L2L3

L2H

7H

6L1

1H

27, L

1H

17H

31

H2

H28

H5

H14

H15

H16

H29

L7L8

H18

H19

(N

-gly

cosy

late

d)

LysC

H1

L5

H8H

9H

8Ly

sC

H4

Minutes10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00

Reduction, alkylation, Lys-C or trypsin digestion

PNGaseF digestion, 2-AB labeling

L Chain Fd’scFc

FabRICATOR digestion, reduction

or reduction (alkylation)

SEC/UV/MS C4 RP-HPLC/UV/MS C18 RP-HPLC/UV/MS HILIC/FLR/MS (100% seq. coverage) (≤100% seq. coverage) (Shang et al.,

J Pharm Sci, 2014)

Creating a mAb CDR Hotspot Database

41

Structural and Computational Biology Team

Elaine Stephens Amy King James Apgar Olga Friese Roger Theberge Marisa Thompson Thomas Powers Michelle English Lisa Marzilli Claire Hendershot Kathleen Cornelius Andrew Saati Leah Wang Samara Ahmed Ying Zhang Robert Kutlik Mellisa Ly Christine Feulner Brian Gau Laura Lin Dennis Gessmann Eric Bennett Andrew Dawdy Meg Ruesch Jason Rouse Will Somers

mAb CDR Degradation Hotspots

Pfizer Confidential │ 42

• Asn and Gln deamidation – Proceeds more quickly if Asn / Gln is followed by a small, flexible residue like glycine - low

steric hindrance leaves peptide group open for attack • Deamidation proceeds more quickly at elevated pH (7-10) and temperature

– Asn / Gln hotspots are molecule and location dependent, and less predictable • Motifs: Primary-NG/QG, NS; Secondary-NT, NY, ND, NW, NQ, NK, NN; Tertiary-NH, NF, NA, NC • L chain hotspots: Asn 30/31 (CDR1, no impact to antigen binding), Asn 92 (CDR3) • H chain hotspots: Asn 55 (CDR2) – can result in decreased antigen binding affinity

• Asp and Glu isomerization – Similar to Asn deamidation in terms of motifs/mechanism, but prevalent at lower pHs (4-7)

• Motifs: Primary - DG/EG; Secondary - DY, DD; Tertiary - DS, DT, DH, DW, DR, DN, DA • H chain hotspots: Asp102 (CDR3) – can result in decreased antigen binding affinity

• Met and Trp oxidation – Surface exposed residues are susceptible to production, analysis & storage conditions

• Hotspots: L chain Met 4; H chain Met 100c (CDR3) • Lys (and Arg) glycation

– Reversible modification that results from high levels of glucose in culture medium • L chain hotspots: Lys 49 (CDR2) • H chain hotspots: Lys 33 (CDR1), Lys 62 (CDR2), Lys 65 (CDR2), Lys 98 (CDR3)

• Polypeptide cleavages: Asp-Pro, Ser-Ser (and Asp-X after deamidation) – Unique peptide bonds that are sensitive to production, analysis, and storage conditions

Sharma et al. PNAS 2014, 111:52, 18601–18606. Haberger et al. mAbs 2014, 6:2, 327–339.

Sydow et al. PLOS ONE 2014, 9:6, e100736.

Creating a mAb CDR Hotspot Database …groundwork for in silico prediction of sequence liabilities

43

99 100 101 102 103 104 105 106 107 108 109

37˚C 4 wks 30.0% 13.0%

3.3% 9.5%

25˚C 4 wks

T=0 0.7 % 2.1%

Asn Asp isoAsp

25SSQSLVHSNGNTFLYWYLQKPGQSPQLLIYR55

…to separate, identify & quantify hotspots

Use the latest LC-MS/MS technologies… Reduce

(DTT) Alkylate

(IAA) Desalt

(BioSpin6) Trypsin/Lys-C

(pH 8.2, 37C)

Orbitrap Fusion (CID and ETD)

Low-artifact digests

0

2

4

6

0 1 2

pH8.2

% d

eam

idat

ion

h h High

selectivity

…to develop a structure-based sequence liability method to predict deamidation/isomerization sites

Compile hotspot data, and share across orgs

mAb1 L Chain CDR Hotspot Database Rel. Abund.

1 DIVMTQTPLSLSVTPGQPASISCRSSQSLV 30 High (>5%) 31 HSNGNTFLYWYLQKPGQSPQLLIYRVSNRF 60 Low (1-5%) 61 SGVPDRFSGSGSGTDFTLKISRVEAEDVGV 90 Potential 91 YYCFQATHVPWTFGGGTKVEIK Framework

Asn deamidation products

S. Philip E. Stephens



Forced Degradation 40°C for 0, 2w and 4w iCE – Herceptin / Trastuzumab

T0 2W 4W

LC CDR1 LC CDR2 LC CDR3 HC CDR1 HC CDR2 HC CDR3 RASQDVNTAVA SASFLYS QQHYTTPPT FNIKDTYIH RIYPTNGYTRYADSVKG WGGDGFYAMDY

Tris buffer pH 7.5 His buffer pH 5.8 Glu buffer pH 4.5

High pI Method Amy King and BMD Colleagues

Red=High-level Hotspot (>5%) Green=Low-level Hotspot (1-5%) Blue=Potential Hotspot (<1%) Orange=non-CDR Hotspot

For Creation of mAb CDR Hotspot Database… How Many LC-MS/MS Peptide Maps Are Really Needed per mAb?

45 45

Unstressed Material (T=0)

LATD (pH 8.2) 30, 60, 120 min

LATD (pH 6.0) overnight

Tris buffer (pH 7.5)

40C, 4 weeks

LATD (pH 8.2) 30 min


His buffer (pH 5.8)

40C, 4 weeks



Glu buffer (pH 4.5)

40C, 4 weeks


LATD (pH 6.0) overnight St

ress

ed C

ondi

tions

ETD

ETD

ETD

ETD

Total of 10 peptide maps (without ETD) per mAb Marzilli et al. LC/GC 2017;15(1): 6–14.

Herceptin / Trastuzumab L Chain (LATD, pH 6.0)


Material M4 (FW1)

D28V (CDR1)

N30T (CDR1) Asn / Asp / isoAsp / Asu

T=0 control 4w, Tris pH 7.5 4w, His pH 5.8 4w, Glu pH 4.5

Harris et al. 2001 (%) --- --- Sydow et al. 2014 (%) --- ---

ASA (%) --- ---

L Chain CDR Hotspots 1 DIQMTQSPSSLSASVGDRVTITCRASQDVN 30

31 TAVAWYQQKPGKAPKLLIYSASFLYSGVPS 60

61 RFSGSRSGTDFTLTISSLQPEDFATYYCQQ 90

91 HYTTPPTFGQGTKVEIKR


Potential Modifications Asn (N,Q) deamidation

Asp (D) isomerization

Succinimide accumulation

Met/Trp (M,W) oxidation

Lys (K) glycation

Asp-Pro (DP) cleavage site

Elaine Stephens



Material M4 (FW1)

D28V (CDR1)


T=0 control 0.1 ND 4w, Tris pH 7.5 0.3 ND 4w, His pH 5.8 0.2 ND 4w, Glu pH 4.5 0.6 ND

Harris et al. 2001 (%) --- --- ~15% (CEX-HPLC) Sydow et al. 2014 (%) --- --- 11% 24%

ASA (%) --- --- 41.7/---






Potential Modifications Asn (N,Q) deamidation

Asp (D) isomerization

Succinimide accumulation

Met/Trp (M,W) oxidation

Lys (K) glycation


Elaine Stephens



Material M4 (FW1)

D28V (CDR1)


T=0 control 0.1 ND 88.8 / 9.7 / 0.8 / 0.7 4w, Tris pH 7.5 0.3 ND 28.2 / 69.8 / 0.6 / 1.4 4w, His pH 5.8 0.2 ND 75.7 / 12.2 / 2.9 / 9.2 4w, Glu pH 4.5 0.6 ND 89.2 / 2.7 / 2.3 / 5.8

Harris et al. 2001 (%) --- --- ~15% (CEX-HPLC) Sydow et al. 2014 (%) --- --- 11% 24%

ASA (%) --- --- 41.7/---






Potential Modifications Asn (N,Q) deamidation: D/E/isoX

Asp (D) isomerization: isoD/D

Asn/Asp succinimide accumulation

Met/Trp (M,W) oxidation: M/W+Ox

Lys (K) glycation: Lys+hexose


88.8

9.7 0.8 0.7 LATD pH 6: t=0

AsnAspisoAspSucc

75.7

12.2 2.9 9.2

LATD pH 6: His buffer AsnAspisoAspSucc

Elaine Stephens

CDR Hotspot Distribution (as of Aug. 25, 2018)

0

16 20 10

1 7

34 27

32 27

10 11

27

71

24 32

24 20

38

132

53 57

25

48

0

20

40

60

80

100

120

140

H-CDR1 H-CDR2 H-CDR3 L-CDR1 L-CDR2 L-CDR3

Hot-spot (>5%)Low-spot (1-5%)Residual (<1%)Not detected

49 Red=High-level Hotspot (>5%) Green=Low-level Hotspot (1-5%) Blue=Potential Hotspot (<1%) Underlined=Not Detected

59 mAbs in database

7% 0% 16% 8% 2% 8%

Heavy Chain Light Chain

mAb CDR Hotspot Database Trends ...as of Aug. 25, 2018

C.XXSQSLXXTD33GTTXXX

C.XASQSVXED32GDSXXXW

C.RXSQSLXXSN33GNTXXX C.RXXXSLXXFN33GNTXXX C.RXSQSIXXSN33GNTXXX

C.XASQXIN30KHXXW

C.XASXSVXHN32GWXXXX

C.RASQXVN30TAXX

C.RXSQSLAN31SYXNTXXX

C.RASQSIN30SYXXW

L-CDR1 H-CDR2

WVA.XIXXKSIN57SAXHY62AEXXKG

WIG.XIXXGN55GDXXYN61QKFKG

WMG.XIXXYN55GNXXYA61RMLXG

WIG.XIXXYN55GGXXYN61QKFKXR

WVG.XIXXYT55GQXXYAD62DFKG

WIG.XIXXYN55DYXXNN61QDFKG

WMG.XIXXET55DEXXYAD62DFKG

WVS.XVXWN54GGXXXYXA62SVKG WVA.XMXYD54GSXXXYXD62SVKG

WIA.XIXXND55DEXXNXA62LKX WVA.XIXXTN55GYXXYXD62SVKG

C.RXSQSIXXXN33GNTXXX WVA.XIXXNN55GDXXYT61QKFKG

Red=High-level Hotspot (>5%) Green=Low-level Hotspot (1-5%) Blue=Trace-level Hotspot (<1%) Underlined=Not detected


Motif L-CDR1 L-CDR2 L-CDR3 H-CDR1 H-CDR2 H-CDR3 PFE%

NG 4(5) --- --- --- 5(7) --- 9/12=75

NS 2(3) 0(1) 0(4) --- 1(4) 1(2) 4/14=29

NT 1(6) 0(1) 0(2) --- 0(12) --- 1/21=5

NY 0(10) --- 0(2) 0(5) 0(9) 2(7) 2/33=6

NF 0(3) --- --- 0(3) 0(2) 0(1) 0/9

ND 0(1) --- 0(1) --- 1(3) --- 1/5=20

NW 0(9) --- 1(4) 0(13) 0(1) 0(1) 1/28=4

NK 1(1) 0(1) --- --- 0(5) --- 1/7=14

NQ 0(1) 0(1) 0(1) --- 1(6) --- 1/9=11

NH --- --- --- 0(1) --- --- 0/1

NN 0(2) 0(2) 0(1) --- 0(5) --- 0/10

PFE% 8/41=20 0/6 1/15=7 0/22 8/54=15 3/11=27

mAb CDR Asn-Deamidation Hotspots (>5%) PFE Database (as of Aug. 25, 2018)


59 mAbs in database

Motif L-CDR1 L-CDR2 L-CDR3 H-CDR1 H-CDR2 H-CDR3 PFE%

DG 2(2) 1(1) --- --- 1(5) 4(7) 8/13=62

DS 0(4) 0(1) 0(1) 0(1) 0(17) 2(2) 2/26=8

DT --- 0(1) --- 0(2) 0(5) --- 0/8

DY 0(1) --- 0(2) 0(7) 0(9) 3(31) 3/50=6

DD --- --- --- 0(2) 3(7) 0(2) 3/11=27

DH 0(2) --- --- 0(1) 0(1) 0(1) 0/5

DW 0(2) --- --- 0(1) --- 0(3) 0/6

DR --- --- --- --- 0(2) 0(4) 0/6

DN 0(3) --- 0(1) --- 0(2) --- 0/6

PFE% 2/14=14 1/3=33 0/4 0/14 4/48=8 9/50=18

mAb CDR Asp-Isomerization Hotspots (>5%) PFE Database (as of Aug. 25, 2018)


59 mAbs in database

Summary and Lessons Learned • MS is an important characterization tool during molecular design, cell-line

selection, and early process development – Minor “process” changes, if needed, can occur without affecting timelines

• MS supports process scale-up & refinement by monitoring PTM consistency – The structural impact, if any, can be assessed in “real-time”

• MS is an essential element of all comparability & similarity exercises – Visualize the protein isoforms that constitute pre-change & post-change batches – Address effect of manufacturing improvements on product quality at isoform level

• Orthogonal methods/mass balance required to properly elucidate structure • With more projects & less time, need to streamline & evolve continuously

– Keep assessing routine methods and strategies, and adjust course as needed – Keep evaluating new technologies, methods & strategies!

• Adoption of new methods and approaches can take a long time – Multiple colleagues (i.e., whole lab/department) needs to reproduce results

• Feedback on method is needed from all colleagues to create all-encompassing, bullet-proof protocol – Takes time to sort out best approach – proactively solicit feedback & ideas

• Enhanced information quality combined w/ efficiencies for better decision making – Many times, the core technology is not ready – need to collaborate with vendors

Continued Partnership between Industry and Hardware/ Software Vendors will Accelerate Future MS Technologies • Software for truly automated deconvolution and MS data analysis

(that eliminates the need for analyst to cross-check data) – LC/MS – / LC-MS/MS – peptide mapping (and MAM) – LC/MS – / LC-MS/MS – glycan mapping

• Including automated differentiation of isomers – LC/MS – / LC-MS/MS – protein/subunit mapping

• Including automated top-down analysis for identification & site-specific mods

• More robust & reliable instruments & intuitive software (MAM) • Commercial solutions for automated sample preparation (MAM) • More efficient, automated higher-order structure MS assessments • Future of mass spec looks bright in biotech!

– Transition from LC / CE profile-based product quality assessments to simultaneously quantitating multiple attributes via LC/MS MAM [1]

– Shift to real-time quality attribute control [2] to ensure greater product consistency and manufacturing efficiency

• Real-time bioreactor analysis will alleviate sample submissions & testing [1] Rogers et al. mAbs 2015, 7, 881-890. [2] Zupke et al. Biotechnol. Prog. 2015, 31, 1433–1441.

Automated Analysis (5-10 min)

5% Manual Analysis (hours!)

Acknowledgements • College of Wooster

– Charles L. Borders – Richard H. Bromund

• Michigan State U – John Allison – Jack T. Watson – Karen L. Wahl – Jeff Gilbert – Gary Schultz – Dave Wagner – Doug Gage – Kurt Kneen – Ed Townsend – Kris Kurtz – ...and many more!

• Genetics Institute / Wyeth / Pfizer – Hubie Scoble – Steve Martin – Jim Vath – Wen Yu – Anne Marie Strang – Marta Czupryn – Tom Porter – Mike Jankowski – Himakshi Patel – Suman (Rathore) Shanker – Smita Karnik – Lisa Marzilli – Meg Ruesch – ...and many more!

• CASSS – Karen Bertani Rohin Mhatre (Biogen) – Renee Olson All committee members!

Acknowledgements • Bruker Daltonics

– Victor Fursey – Brian Stall – Paul Kowalski (Waters) – Doug Roderick – Terry Murphy – ...and many more!

• Waters – Robin Andreotti – Roy Martin – Weibin Chen – Scott Berger – Ian Lloyd – Don Kwet – ..and many more!

• New Objective – Gary Valaskovic

• ProtTech – Drake Zhang

• SGS M-Scan – Mark Rogers – Chris Ziegenfuss (BioPharmaSpec) – Scott Paris

• Protein Metrics – Eric Carlson

• Just Biotherapeutics – Rich Rogers

• Genentech – Chris Yu

• Thermo Scientific – Jonathan Josephs – Jennifer Sutton – Aaron Bailey – Stephane Houel – Betty Woo – Dave Jarzinski – ..and many more!

Acknowledgements

MSBC-PCG Cross-site Technical Exchange Meeting, June 2017, Lake Forest, IL

Backup Slides

unraveling the structural heterogeneity of recombinant ... · – andy hanneman (charles river) ......

Documents