1© 2017 USP

USP Standards to Support Multi-Attribute Methods (MAM) and Mass Spectrometry

Diane McCarthy, Ph.D. Global Biologics

January 10, 2020

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Outline

Standards to Support Mass Spectrometry

USP MAM Efforts

Monoclonal Antibody Standards

– Characterization

– MAM method development

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Intact monoclonal antibody standards– Multiple standards with different properties

– Applicable for system suitability, training, tech transfer, method development

– Analysis of modifications by peptide mapping and orthogonal methods (cIEF, CEX, released glycan, etc.)

Pre-digested monoclonal antibody standard– Suggestion from Mass Spec Peptides working group

– Proposed uses include system suitability, instrument qualification, tech transfer, and training and proficiency testing

Host cell protein standards– Standards to support identification and quantitation of individual problematic HCPs

– Full length protein and/or corresponding peptides

Mass Spectrometry Standards under DevelopmentIdeas from Mass Spec Peptides Working Group, Expert Committees, Roundtables

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USP is exploring MAM as:– A method to provide more efficient and comprehensive protein characterization of USP

Reference Standards– A means to select an appropriate material for a pre-digested mAb standard– An area where documentary and/or physical standards may be useful

Conducted a small survey to understand current practices for MAM/ peptide mapping

Collaborating with University of Georgia’s Complex Carbohydrate Research Center to develop MAM methods– Initial focus on mAbs– Expect to extend to other proteins

Multi-Attribute Methods (MAM) at USP

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Trypsin LysC Trypsin/LysC(combo)

GluC AspN CPB, Ides

Results of USP Survey on MAM

Modifications Monitored Digestion Enzyme

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Results of USP Survey on MAM (cont.)

Use of New Peak Detection

Monoclonalantibody reference

standards

Other protein (non-mAb) reference

standards

Other referencestandards

Not usingstandards

Sometimes - not withevery analysis

Routinely - with everyanalysis

Never

Reference Standards

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Characterization of Monoclonal Antibody Standards

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USP plans to release 3 new mAbs – Stand alone reference standards – not associated with a monograph/chapter– Include characterization package for each one– Customers can choose which mAb best meets their needs based on characteristics

Initial Characterization Package will include:– <129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies

• SEC• CE-SDS (reducing and non-reducing)• Oligosaccharide Analysis• Sialic Acid Analysis

– Intact mass (by mass spectrometry)– Oligosaccharides by HILIC-FLD-MS

Plans for New mAb Materials

Other Applications under Exploration− cIEF− CEX− SEC-MALS

− CE-MS− Fc binding− Column qualification

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mAb001 mAb002 mAb003

N-terminal Pyroglutamate (LC)

92.6% (Q→pE) NA (N-terminal is other amino acid) NA (N-terminal is other amino acid)

N-terminal Pyroglutamate (HC)

99.9% (Q→pE) 0.8% (E→pE) 1.5% (E→pE)

C-terminal Lys deletion (HC)

89.2% 98.3% 82.3%

Deamidation (LC) (>3%are listed)

3.7%(~N136), 10.2%(~N151), 4.8%(Q3/Q6), 9.2%(Q27 /N31/N35), 7.7%(N152 / Q155/N158/Q160/Q166)

5.6%(Q5/Q15), 3.8%(Q35/Q36), 23.0%(Q40/N50/N51), 16.8%(N67/Q77/N87/N94),

Deamidation (HC) (>3%are listed)

3.2%(N33), 66.5%(~N55), 13.7%(~N163), 14.5%(~N280), 41.8%(~N319), 36.2%(~N388), 9.4%(~Q423)

6.8%(Q3/Q13), 3.7%(N31/N35), 3.5%(N52), 27.7%(Q82/N84),4.9%(N282/N292), 42%(Q317/N321), 26.7%(N390/Q392/N395/N396)

4.8%(Q3/Q6), 8.8%(N74), 23.2%(N77/N84), 14.9%(N163/Q179/Q200/N205/N207/N212), 6.3%(N280/N290), 43.2%(Q315/Q319), 28.4%(N388/Q390/N393/N394), 10.6%(Q422/Q423/N425)

Oxidation (LC) Not observed 1.2%(M4) Not observed

Oxidation (HC) 0.6%(M34), 0.7%(M37), 1.1%(W162), 9.6%(M256),

3.7%(M34), 0.7%(M83), 5.4%(M258), 2.1%(M364)

3.3 %(M34), 0.3%(W47),6.6%(M256), 2.2%(M432)

Summary of PTMs identified in peptide mapping

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Characterization of mAbs using MAM

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Post-Translational Modifications– Deamidation– Oxidation– N- and C-terminal clipping– Pyroglutamate– Glycosylation– Glycation– Phosphorylation– Sulfation– Methylation– Acetylation– Hydroxylation

Workflow and Target PTMs

Whole Protein

Reduced Protein

Reduction, Alkylation

Protease Digestion (Trypsin)

Peptides

C18 Clean up/Desalting

LC-MS/MSC18 Separation MS and MS/MS (DDA)

Data AnalysisByonic™ and/or Proteome Discoverer™

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Composition Alternate Nomenclature USP mAb001 USP mAb002 USP mAb003

HexNAc(4)Hex(5)Fuc(1)NeuAc(1) G2FS1 0.81% 0.45% 1.44%

HexNAc(4)Hex(5)Fuc(1) G2F 7.15% 2.69% 5.93%HexNAc(4)Hex(4)Fuc(1) G1F 43.82% 30.16% 38.47%HexNAc(4)Hex(3)Fuc(1) G0F 41.60% 58.68% 44.62%HexNAc(4)Hex(4) G1 0.28% 0.11% 0.19%

HexNAc(4)Hex(3) G0 0.56% 0.13% 0.72%

HexNAc(3)Hex(5) Hybrid 0.17% 0.09% 0.18%

HexNAc(3)Hex(3)Fuc(1) G0F-N 2.97% 3.76% 5.19%HexNAc(3)Hex(3) G0-N 0.91% 0.57% 2.52%

HexNAc(2)Hex(9) Man 9 0.04% 0.01% 0.00%

HexNAc(2)Hex(8) Man8 0.78% 0.33% 0.22%

HexNAc(2)Hex(7) Man7 0.37% 0.37% 0.06%

HexNAc(2)Hex(6) Man6 0.01% 0.15% 0.03%

HexNAc(2)Hex(5) Man5 0.01% 0.02% 0.02%

HexNAc(2)Hex(4) Man4 0.02% 0.54% 0.04%

HexNAc(2)Hex(3)Fuc(1) 0.01% 0.39% 0.02%

HexNAc(2)Hex(3) 0.25% 0.76% 0.20%

HexNAc(1)Fuc(1) 0.11% 0.26% 0.07%

HexNAc(1) 0.13% 0.52% 0.06%

Relative Percentage of Glycoforms on Peptide EEQYNSTYR

Glycopeptides were initially identified using Byonic™ software, which requires confirmation by MS/MS

As a supplemental analysis, a manual search was also performed

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MAM Method Also Yields High Deamidation

Deamidation rates remained high using MAM method

Similar results using two platform methods suggests materials may be especially susceptible – Consequence of age, storage?

Additional work needed to assess source of deamidation and distinguish between– Artifact of sample preparation

– Inherent property of mAb

Characterization (contract lab)

MAM Results (CCRC)

DTLMISR 418.2203 2 --- ---

DTLmISR 426.217 2 Oxidation 9.60% 9.80%

FNWYVDGVEVHNAK 559.9388 3 --- ---

FnWYVDGVEVHNAK 560.2681 3 Deamidation 14.50% 6.60%

FNwYVDGVEVHNAK 847.4009 2 Oxidation ND* 0.10%

VVSVLTVLHQDWLNGK 603.3403 3 --- ---

VVSVLTVLHQDWLnGK 603.6705 3 Deamidation 41.80% 28.70%

VVSVLTVLHQDwLNGK 608.6711 3 Oxidation 0.04%

NQVSLTCLVK 552.8077 2 --- ---

nQVSLTCLVK 553.299 2 Deamidation ND 9.10%

GFYPSDIAVEWESNGQPENNYK 848.7149 3 --- ---

GFYPSDIAVEWESnGQPENNYK 849.0495 3 Deamidation 36.20% 10.40%

WQQGNVFSCSVMHEALHNHYTQK 915.4193 3 --- ---

WQqGNVFSCSVMHEALHNHYTQK 915.7486 3 Deamidation 9.40% 8.20%

WQQGNVFSCSVmHEALHNHYTQK 920.7517 3 Oxidation ND 1.90%

Peptide Sequence m/z z ModificationRelative % of Modification

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Optimization of Peptide Mapping Methods (Round 1)

Buffer exchange into 50mM ammonium bicarbonate

10 kDa MWCO

Add Rapigest to 0.1%

Add Trypsin and Digest overnight at 37C

Stop Trypsin (heating)Reduce with DTT 60C for 30

minutes

Acidification by TFA to remove Rapigest

Dissolve sample in 50mM Tris-Cl pH 8; Add 5mM DTT and incubate

for 45 minute at 60C

Add 13 mM IodoacetamideIncubate at RT for 45 minutes

Add another 5mM DTT. Incubate at RT for 15 minutes

Cool and add Trypsin 1:50. Incubate overnight at 37C

Inject for LC-MS/MS

Initial Bulk Characterization Initial MAM Method

[1] Anal Chem. 2009, 81, 1686-1692

Mix protein with 0.1% RG at 1:1 (w:w) in 50mM Tris-Bis pH 6.0

Add 5X TCEP to protein (molar ratio)(0.3mM)

Incubate 60C for 30 minutes

Cool and add Trypsin 1:50Incubate 2hr at 37C

Quench reaction and decompose RG with 25% TFA. Centrifuge to remove

RG and supernatant for analysis

Low pH Digestion1

2 hr at 37C

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Comparison of Deamidation and Oxidation with Different Digestion Methods

Contract Lab (MS only)

MAM w/ ON digestion

MAM w/ 2 hr digestion

MAM w/2 hr low pH

digestionDTLMISR 418.2203 2 --- ---

DTLmISR 426.217 2 Oxidation 9.60% 9.80% 5.60% 5.80%

FNWYVDGVEVHNAK 559.9388 3 --- ---

FnWYVDGVEVHNAK 560.2681 3 Deamidation 14.50% 6.60% ND ND

FNwYVDGVEVHNAK 847.4009 2 Oxidation ND* 0.10% 0.20% 0.30%

VVSVLTVLHQDWLNGK 603.3403 3 --- ---

VVSVLTVLHQDWLnGK 603.6705 3 Deamidation 41.80% 28.70% ND **

VVSVLTVLHQDwLNGK 608.6711 3 Oxidation 0.04% ND **

NQVSLTCLVK 552.8077 2 --- ---

nQVSLTCLVK 553.299 2 Deamidation ND 9.10% ND ND

GFYPSDIAVEWESNGQPENNYK 848.7149 3 --- ---

GFYPSDIAVEWESnGQPENNYK 849.0495 3 Deamidation 36.20% 10.40% 2.80% 6.00%

WQQGNVFSCSVMHEALHNHYTQK 915.4193 3 --- ---

WQqGNVFSCSVMHEALHNHYTQK 915.7486 3 Deamidation 9.40% 8.20% ND **

WQQGNVFSCSVmHEALHNHYTQK 920.7517 3 Oxidation ND 1.90% 1.70% **

Peptide Sequence m/z z Modification

Relative % of Modification

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Comparison of 4 digestion methods showed– Shorter digestion time and/or low pH reduced deamidation rates

– Oxidation was also reduced in several cases

– However, both methods with 2 hr digestion time showed more incomplete digestion

Further optimization is necessary to improve digestion efficiency while still reducing artifacts

Additional work is underway to assess impact of– Increasing trypsin concentration

– Guanidine HCl

Selected method will then be tested on other mAbs and complex glycoproteins– Expect some optimization will be needed for specific molecules

Results and Next Steps

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diane.mccarthy@usp.org