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
Fast desalt on HRAM MS [10mins]
- 0.5 to 1ug
- 1 x 50mm column
Data analysis and report generation
- Protein deconvolution [LC & HC]
- Assignment of putative glycoforms on heavy chain
4. Partial Digest [IdeS, SpeB]
IdeS Digestion
Fast desalt on HRAM MS [10mins]
- 0.5 to 1ug
- 1 x 50mm column
Data analysis and report generation
- Protein deconvolution [Fc,
Fd, F(ab)2]
PNGaseF Digestion
Fast desalt on HRAM MS [10mins]
- 1 to 2ug
- 1 x 50mm column
Data analysis and report generation
- 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
Time [min] % Solvent B Valve Position
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
Time [min] % Solvent B Valve Position
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 2 + DTT + PNGaseF
IgG Std 1 & Std 2 – Heavy Chain
IgG Std 1 + DTT
IgG Std 1 + DTT + PNGaseF
IgG Std 2 + DTT
IgG Std 2 + DTT + PNGaseF
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-
Time [min] % Solvent B Valve Position
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-
Time [min] % Solvent B Valve Position
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
23,788.38 [+ sugars] 23,419.77 [+ sugars] 25,112.85 [+ sugars]
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
24,865.98 [+ sugars] 23,419.32 [+ sugars] 24,034.17 [+ sugars]
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