protein therapeutics: lc-ms based bioanalytics · pipeline of protein therapeutics entering, or in,...

Protein Therapeutics: LC-MS Based Bioanalytics

Antibody Characterization Workflows from a CRO

Perspective

Presented by: Greg W. Kilby, Ph.D [email protected]

Introduction to Protea Biosciences

- Based in Morgantown, WV

- ~11,000 sq. ft. state-of-the-art services laboratory

- Provider of MS consumables, technologies, and services

- Customers from Pharma, Biopharma, CROs, Consumer

Products, Medical and Life Sciences industries.

- Focused on mass spectrometry based identification,

quantification, characterization and distribution of biomolecules

[proteins, metabolites, and lipids] from biological matrices.

Introduction There is an expanding inventory of FDA approved biotherapeutics currently on the market and a rapidly growing pipeline of protein therapeutics entering, or in, clinical trials, that are based around monoclonal antibodies (mAb’s). mAb’s produced from mammalian cells may be present in multiple isoforms due to a variety of mechanisms, such as alternate splicing, post-translational modifications (PTM’s), and variations in genetic code. These variations can result in different biological consequences. As such, it is highly desirable to identify and characterize mAb’s to ensure the quality, safety, and efficacy of these therapeutics. Although large portions of human IgG’s are highly conserved, each mAb drug product can display significant heterogeneity due to designed attributes, differences in manufacturing processes, and under storage conditions. Human IgG’s are subject to a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, and terminal cyclization. The detailed analysis of post-translational modifications (PTMs) present on mAb’s is required to ensure both safety and efficacy. Due to the [sometimes extreme] heterogeneity of mAb’s, thorough characterization still remains challenging. We present here a comprehensive antibody characterization workflow, coupling capillary HPLC with an Agilent 6530 High-Resolution/Accurate-Mass (HR/AM) Quadrupole Time-of-Flight mass spectrometer.

Monoclonal Antibodies 101

• Monoclonal antibodies (mAbs) • Monospecific → from identical immune cells, clones of a unique parent cell • Bind to the same epitope [monovalent affinity] • Large heterodimeric molecules [~150kDa]

• The majority of marketed mAbs therapeutics → IgG class • 4 subclasses – IgG1 through IgG4 • Common drug name ends in mab • Bulk of “mab” therapeutics are targeted at inflammation or oncology

• IgG’s • 2 identical HC and 2 identical LC • 16 inter- or intra-molecular disulfide bonds. • IgG’s include antigen-binding (Fab) and crystallizable (Fc) regions • Fc binds to Fcγ receptors, which regulate immune responses.

• LC and HC • Class of Ig is determined by type of HC

• Gamma-1 HC → IgG1, Gamma-2 HC → IgG2 up to IgG4 • 2 types of LC; kappa and lambda [but not both]

• Therefore IgG1 → γ12λ2 or → γ12κ2

Areas of Interest in mAb Analysis

• Glycoform variability mainly relating to heterogeneous N-linked glycan populations → mediates biological activity

• safety, efficacy, stability • Charge variants resulting from several types of protein modifications Amino acid modifications such as Asparagine deamidation, Aspartate isomerization, and formation of N-terminal pyroglutamate

• physiochemical and functional stability • Cysteine-related variants and oxidation states • Molecular mass and primary amino acid variants including C-terminal

lysine clipping • Secondary through quaternary protein structure and antigen complex

variations • Dimerization and aggregation • Contamination with host cell proteins [manufacturing]

Anatomy of an Antibody – Human IgG

Cys229

Cys232

CH3

C

H2

Cys264

Cys324

Cys370

Cys428

CH3

Cys370

Cys428

Carbohydrate N300

CH2

Cys264

Cys324

5

1

2

6

3

4

1. F(ab’)2 • Fab x 2 = Fd + LC • Binds antigen

2. Fc • scFc x 2 • crystallizable

3. HC • VH+CH1+Hinge+CH2+CH3

4. LC • VL+ CL1

5. Antigen Binding • VL + VH

• 3 hyper variable regions each • CDR – complementarity

determining regions 6. Hinge

• Contains IdeS cleavage site • …CPPCPAPELLG239 / G240PSVF…

Code Glycan Structure Mono Mass [Da] Ave Mass [Da]

G0 1298.4760 1299.18

GOF 1444.5339 1445.32

G1F 1606.5867 1607.46

G2F 1768.6395 1769.60

Galactose 162.0528 162.1424

Mannose 162.0528 162.1424

GlcNac 203.0794 203.1950

Fucose-1,6 146.0579 146.1430

Typical Carbohydrate Structures of Human IgG

Minimal sample prep.

- dilution

- buffer exchange

Fast desalt on HRAM MS [10mins]

- 1 to 2ug

- 1 x 50mm column

Data analysis and report generation

- Protein deconvolution

- Assignment of putative glycoforms on native mAb

1. Intact M.Wt Confirmation

3. Reduced [LC & HC]

Reduce

- DTT or TCEP

- Alkylate If proceeding to peptide map or disulfide

mapping


- 0.5 to 1ug

- 1 x 50mm column


- Protein deconvolution [LC & HC]

- Assignment of putative glycoforms on heavy chain

4. Partial Digest [IdeS, SpeB]

IdeS Digestion


- 0.5 to 1ug

- 1 x 50mm column


- Protein deconvolution [Fc,

Fd, F(ab)2]

PNGaseF Digestion


- 1 to 2ug

- 1 x 50mm column


- Protein deconvolution

- Naked mAb M.Wt.

2. Deglycosylation [PNGaseF]

Typical Workflows for Analysis of Intact mAb’s

From a CRO perspective

What do our sponsors want?

• Access to technology AND expertise

• A collaborative approach

• Capacity & scalability

• Sensitivity*

LC-MS Analysis of Intact mAb’s

HPLC Conditions: Column – Poroshell SB300 C8 75 x 0.5mm Column – Poroshell SB300 C3 100 x 1.0mm Column Temp – 60°C AS Temp – 4°C Flow – 100mL/min Solvent A – 0.1% aq. Formic Acid Solvent B – ACN containing 0.1% Formic Acid

Time [min] % Solvent B Valve Position

5 5 To Waste

15 90 To MS

16 90 To MS

16.5 5 To MS

MS Conditions: Time Segment 1 : 0 to 5min, divert valve to waste, not collecting MS data Time Segment 2 : 5min to As Pump, divert valve to MS, collecting Profile data Source – Dual ESI, 1 point reference correction, 922.0098 MS Range – 500 to 5000m/z at 0.5sec/scan Gas temp – 350°C Gas Flow – 10L/min Nebulizer – 25psig Vcap – 3500V Fragmentor – 350V

Common for all intact and sub unit domain analysis

TIC IgG Std 1 TIC IgG Std 2

2mg on C3

2mg on C8

2mg on C3

2mg on C8

Every Antibody is Different……

MaxEnt Deconv. IgG Std 1 MaxEnt Deconv. IgG Std 2

Frag 400V

Frag 350V

Frag 400V

Frag 350V

15ppm

4ppm

Reproducibility – 10 replicate injections overlaid

IgG Std 1

IgG Std 1 – semi-manual interpretation [sequence is known]

G0F/G0F

G1F/G0F

G1F/G1F

G2F/G1F

G2F/G2F

IgG Std 2 – semi-manual interpretation [sequence is unknown]

P1 P2

P3

P4 P5 P6

P7

Automation of Data Analysis and Reporting

Leave out if sequence is unknown

Customizable Reports

IgG Std 1 Mass Accuracy Stability [Sequence is Known]

IgG Std 2 Mass Accuracy Stability [Sequence is Not Known]

Deglycosylation using PNGaseF N-Linked Glycans

- poroshell c8, 75mm x 500um, 60°C, 100µL/min - Amidase from Flavobacterium meningosepticum

- Cleaves at GlcNAc and Asn if Fucose is a[1-6] - Incubate 25k unit/µg of protein for 4 hrs at 37°C

- Vcap – 3500V - Fragmentor – 350V

Cys229

Cys232

CH3

C

H2

Cys264

Cys324

Cys370

Cys428

CH3

Cys370

Cys428

Carbohydrate N300

CH2

Cys264

Cys324

5

1

2

6

3

4


5 5 To Waste

15 90 To MS

16 90 To MS

16.5 5 To MS

IgG Std 1 – Incubation with PNGaseF

IgG Std 2 – Incubation with PNGaseF

PNGaseF

Native

2889.3 [G0F/G0F]

Sub Unit Domain Analysis Mild Reduction → LC & HC

Mild Reduction + PNGaseF → LC & HC (naked?)

- poroshell c8, 75mm x 500um, 60°C, 100uL/min

- 5mM DTT at 37°C for 60 min

- Vcap – 3.5kV

- Fragmentor – 250V

Cys229

Cys232

CH3

CH2

Cys264

Cys324

Cys370

Cys428

CH3 Cys370

Cys428

Carbohydrate

N300

CH2

Cys264

Cys324

5

1

2

6

3

4


4.5 5 To Waste

5 10 To MS

15 50 To MS

15.5 90 To MS

16.5 90 To MS

17 5 To Waste

Reduced IgG Std 1 & 2 – [DTT + PNGaseF]

LC

HC IgG Std 1 - TIC

IgG Std 2 - TIC LC HC

IgG Std 1 & Std 2 – Light Chain

IgG Std 1 + DTT

IgG Std 1 + DTT + PNGaseF

IgG Std 2 + DTT


IgG Std 1 & Std 2 – Heavy Chain

IgG Std 1 + DTT


IgG Std 2 + DTT


Sub Unit Domain Analysis IdeS [limited]Digestion → scFc & F(ab’)2

IdeS + DTT → scFc & Fd & LC PNGaseF +IdeS + DTT→ scFc [naked] & Fd & LC

- poroshell c8, 75mm x 500um, 60°C, 100uL/min - Cysteine Protease, S. Pyogenes

- Cleaves below the hinge region - 1 unit of IdeS per 1 µg of protein

- incubate at 37°C for 30 min - 5mM DTT at 37°C for 60 min

- Vcap – 3.5kV - Fragmentor – 250V

Cys229

Cys232

CH3

C

H2

Cys264

Cys324

Cys370

Cys428

CH3

Cys370

Cys428

Carbohydrate N300

CH2

Cys264

Cys324

5

1

2

6

3

4

-CPPCPAPELLG / GPSVF-


4.5 5 To Waste

5 10 To MS

15 50 To MS

15.5 90 To MS

16.5 90 To MS

17 5 To Waste

Best Option for Middle Down

IgG Std 1 & 2 – IdeS “Limited” Digestion [+ PNGaseF]

F(ab’)2

scFc IgG Std 1

F(ab’)2

scFc IgG Std 2

IgG Std 1 & 2 – scFc

G0F G1F

G2F -GlcNAc

1444.56 Da

IgG Std 1 - IdeS

IgG Std 1 - IdeS + PNGaseF

IgG Std 2 - IdeS

IgG Std 2 - IdeS + PNGaseF

1444.46 da

IgG Std 2 – IdeS “Limited” Digestion – F(ab’)2

IdeS

IdeS + PNGaseF

1445.6

+scFc [25,218 +H2O = 25,236]

scFc

LC Fd

scFc

LC

Fd

IgG Std 1 – IdeS + PNGaseF + DTT

scFc LC

Fd

scFc

LC

Fd

IgG Std 2 – IdeS + PNGaseF + DTT

Sub Unit Domain Analysis SpeB + DTT→ scFc & Fd & LC

- poroshell c8, 75mm x 500um, 60°C, 100uL/min - Recombinant Cysteine Protease, S. Pyogenes

- Cleaves above hinge, requires reducing conditions - 1ug SpeB/ug protein, 5mM DTT, 37°C for 1hr

- Vcap – 3.5kV - Fragmentor – 250V

Cys229

Cys232

CH3

C

H2

Cys264

Cys324

Cys370

Cys428

CH3

Cys370

Cys428

Carbohydrate N300

CH2

Cys264

Cys324

5

1

2

6

3

4

-KTHT / CPPCPAPELLG-


4.5 5 To Waste

5 10 To MS

15 50 To MS

15.5 90 To MS

16.5 90 To MS

17 5 To Waste

IgG Std 1 & Std 2 – SpeB + DTT + PNGaseF

scFc

LC

Fd

scFc/LC

Fd

IgG Std 1 & Std 2 – SpeB + DTT + PNGaseF, MaxEnt Deconvolution

scFc

LC

Fd

scFc

LC

Fd

IgG Std 1

IgG Std 2

mAb Sub Unit Domain Analysis - Summary

IgG Std 1 IgG Std 2

Light Chain +DTT +PNGaseF

23,124.58 23,124.54

23,419.25 [+ sugars] 23419.37 [+ sugars]

Heavy Chain +DTT G0F G1F G2F +PNGaseF

50,900.53 51,062.69 51,224.60 49,455.95

50,326.88 50,488.67 50,650.21 48,883.43 [+ sugars]

mAb Sub Unit Domain Analysis - Summary Subunit IgG Std 1 IgG Std 2

IdeS scFc G0F scFc G1F scFc G2F F(ab’)2 F(ab’)2 + 1xscFc

25,233.23 25,395.32 25,557.33 97,611.79

25,232.86 25,394.96 25,557.03 97,057.42 [+ sugars] 122,275.51 [+ sugars]

IdeS + PNGaseF scFc F(ab’)2 F(ab’)2 + 1xscFc

23,788.67 97,612.46

23,788.40 [+ sugars] 97,058.67 [+ sugars] 120,829.91 [+ sugars]

IdeS + PNGaseF + DTT scFc LC Fd

23,788.36 23,124.33 25,685.81


Note: scFc is clipped below the hinge

mAb Sub Unit Domain Analysis - Summary

Subunit IgG Std 1 IgG Std 2

SpeB + DTT scFc – G0F scFc – G1F scFc – G2F LC Fd

26,310.71 26,472.86 26,634.90 23,124.21 24,607.23

26,310.81 26,472.89 26,634.91 23,419.38 [sugars] 24,034.38 [sugars]

SpeB + PNGaseF + DTT scFc LC Fd

24,866.87 23,124.33 24,607.19


Note: scFc is clipped above the hinge

IgG SpeB IdeS D (Da)

Std 1 scFc 24,866.87 23,788.67 1078.20

Fd 24,607.23 25,685.81 1078.58

scFc+Fd [-H2O] = HC 49,456.10 49,456.48 _

HC [DTT reduction] 49,455.95 49,455.95 _

Std 2 scFc 24,865.98 23,788.38 1077.60

Fd 24,034.17 25,112.85 1078.68

scFc+Fd = HC 48,882.15 48,883.23 _

HC [DTT reduction] 48,883.43 48,883.43 _

-KTHT / CPPCPAPELLG / GPSVF-

1096.34 – H2O = 1078.34

Ave. = 1078.27

Hinge Peptide

Update - From a CRO perspective

• “Industrialized” methodology presented here forms the basis of a bespoke platform method for industry sponsors/partners

• Allows quicker turn around time for small AND large scale projects

• FTE time • On the fly method development • Data analysis, interpretation, and reporting

Multi Enzyme Peptide Map Workflow for Sequence

Characterization of Proteins

Enzymatic Digestion

- Trypsin

- Custom [LysC, GluC, AspN, pepsin, PNGaseF, etc]

- +/- Red & Alk

LC-MS [60mins or longer]

- 5µg on column

- 1 x 150mm [minimum] column

Sequence Coverage PTM Analysis Disulfide mapping

Peptide Quantitation

• Tryp/LysC – C-term Arg & Lys

• Tryp – C-term Arg & Lys [Lys not as efficient]

• LysC – C-term Lys

• Chymotrypsin – C-term Phe, Trp, Tyr [serine proteinase family]

• Pepsin – C-term Ala, Val, Leu, Ile, Phe, Trp, Tyr [hydrophobic AA’s – pH specific]

• AspN – N-term Asp, Cys, Gln

• GluC – C-term Asp, Glu

• Elastase – C-term small, hydrophobic AA’s such as Gly, Ala, Val

• Proteinase K – peptide bond adjacent to the carboxyl group of aliphatic and aromatic AAs

Tryp/LysC

Tryp

LysC

ChymoT

Pepsin

Elastase

IgG Std 1 – Multi enzyme digestions

Enzyme Chain % Seq. Coverage IgG 1 % Seq. Coverage IgG 2*

Tryp/LysC HC LC

96 98

89 47

Tryp HC LC

91 98

78 60

LysC HC LC

89 98

77 42

ChymoT HC LC

61 76

40 37

Pepsin HC LC

84 91

73 59

Elastase HC LC

58 54

66 41

* The peptide map data for IgG Std 2 was searched against the IgG1 Std 1 sequence

Multi enzyme digestions; Sequence Coverage from BioConfirm

HC Sequence of IgG Std 1 - Alignment for IgG Std 2

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTS

RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI

CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT

KNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K Tryp/LysC

Tryp

LysC

pepsin ChymoT Elastase

LC Sequence of IgG Std 1 - Alignment for IgG Std 2

DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

Tryp

LysC pepsin ChymoT

Elastase

Tryp/LysC

Conclusion

• Developed and tested Workflows for thorough LCMS

Based Characterization of mAb’s

• Intact

• Sub Unit Domains

• Multi-enzyme Peptide Maps

• Agilent Q-TOF technology combined with BioConfirm

data analysis workflows is a reliable, robust, easy to

use platform for routine mAb characterization

Thank You !!!

Acknowledgements

Matthew Maust

Kyle Cahill

Devin George

Sponsors [who shall remain anonymous]

Genovis

Agilent technologies

protein therapeutics: lc-ms based bioanalytics · pipeline of protein therapeutics entering, or in,...

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