[ CARE AND USE MANUAL ]

1

CONTENTS

I. INTRODUCTION

II. RECOMMENDED RECONSTITUTION

III. STORAGE AND STABILITY

IV. SEQUENCE AND MASS INFORMATION

V. EXAMPLE RPLC CONDITIONS AND REPRESENTATIVE DATA a. Peptide mapping with formic acid, difluoroacetic acid, and trifluoroacetic acid mobile-phase conditions. b. Routine peptide mapping with the BioAccord System

VI. CAUTIONARY NOTE

VII. ORDERING INFORMATION

I. INTRODUCTIONPeptide level analysis of proteins by LC-MS is one of the primary approaches for the characterization of biopharmaceuticals, in particular monoclonal antibodies (mAbs). In such an analysis, it is possible to acquire peptide mass information, quickly confirm protein sequence and post-translation modifications, as well as degradation related critical quality attributes. To support this application area, Waters has commercialized the mAb Tryptic Digestion Standard, which can be used in the benchmarking of LC-MS techniques, proficiency testing among different instruments and laboratories, and system suitability when performing peptide level protein analysis. This standard is a stabilized and lyophilized format of a reduced and alkylated tryptic digest of NIST Reference Material 8671 (NIST mAb), a humanized IgG1κ expressed from a murine cell line.* It is prepared with trehalose excipients to help ensure stability. Each vial of the standard is provided in an approximately 40 µg quantity.

*Disclaimer: Reference values for RM 8671 are not certified to be in correspondence with any Report of Investigation offered by the National Institute of Standards and Technology (NIST). If it is desirable to have such correspondence, it is advised that RM 8671 be directly acquired from NIST (nist.gov).

mAb Tryptic Digestion Standard

Figure 1. Crystal structure of an intact IgG monoclonal antibody.

2mAb Tryptic Digestion Standard

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II. RECOMMENDED RECONSTITUTIONIt is recommended to dissolve the standard in 80 µL of 0.1% formic acid in 18.2 MΩ water while carefully aspirating or vortexing to mix. Use injection volumes between 2 and 20 µL for 2.1 mm I.D. columns.

For testing with the BioAccord™ System, it is recommended to dissolve the standard in 200 µL of 0.1% formic acid in 18.2 MΩ water and to inject 5 µL onto a 2.1 mm I.D. column. For more information, see Section V.

III. STORAGE AND STABILITYUpon arrival and prior to reconstitution, please store the standard in its original packaging at -20 °C up until its marked expiration date. After reconstitution, the standard can be stored at 4–8 °C for 7 days without concern of degradation.

IV. SEQUENCE AND MASS INFORMATIONOne intact molecule of NIST mAb/RM 8671 is comprised of 2 heavy and 2 light chain subunits covalently linked through 12 intra-chain and 4 inter-chain canonical disulfide bonds. A canonical site of IgG glycosylation is also present in the heavy chain and is marked below in bold. The heavy chain N-terminus is present in the form of a pyroglutamate residue.

The reduced and alkylated tryptic digest of NIST mAb/RM8671 contains no intact disulfide bonds and all cysteine residues have been reduced and capped with one carboxymethyl group. Trypsin cleaves peptide bonds at the carboxyl side of lysine or arginine, except when either of them is followed by proline. All trypsin cleavage sites are marked below in red. It is common nomenclature to define a tryptic peptide with a “T” and to sequentially assign numbers to each theoretical peptide. Accordingly, an LC:T2 peptide label denotes a peptide having a sequence of VTITCSASSR.

Heavy Chain (HC) SequencepQVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Light Chain (LC) Sequence

DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

3mAb Tryptic Digestion Standard

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List of major tryptic digestion peptide from NIST mAbPeptide ID Expected mass (Da) Peptide sequence Modifications*HC:T1 599.3457 pQVTLR Pyroglutamic Acid Q N-TERM

HC:T2 3700.8713ESGPALVKPTQTLTLTCTFSGFSLST AGMSVGWIR

Carbamidomethyl C

HC:T4 1660.8006 ALEWLADIWWDDKHC: T4-5 1788.8956 ALEWLADIWWDDKK Miss-cleavageHC:T8 561.3606 LTISKHC:T10 700.4352 NQVVLKHC:T11 1848.7891 VTNMDPADTATYYCAR Carbamidomethyl CHC:T12 2801.3127 DMIFNFYFDVWGQGTTVTVSSASTKHC:T13 1186.6467 GPSVFPLAPSSKHC:T14 1321.6780 STSGGTAALGCLVK Carbamidomethyl C

HC:T15 6713.2913DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTK

Carbamidomethyl C

HC:T20 2844.4575 THTCPPCPAPELLGGPSVFLFPPKPK Carbamidomethyl C (2)HC:T21 835.4342 DTLMISRHC:T22 2139.0274 TPEVTCVVVDVSHEDPEVK Carbamidomethyl CHC:T23 1677.8020 FNWYVDGVEVHNAKHC:T25 2634.0459 EEQYNSTYR Glycosylation G0FHC:T26 1808.0065 VVSVLTVLHQDWLNGKHC:T30 838.5033 ALPAPIEKHC:T36 1161.6296 NQVSLTCLVK Carbamidomethyl CHC:T37 2544.1314 GFYPSDIAVEWESNGQPENNYKHC:T38 1873.9218 TTPPVLDSDGSFFLYSKHC:T39 575.3399 LTVDKHC:T41 2801.2671 WQQGNVFSCSVMHEALHNHYTQK Carbamidomethyl CLC:T1 1892.9018 DIQMTQSPSTLSASVGDRLC:T2 1081.5306 VTITCSASSR Carbamidomethyl CLC:T3 1621.7944 VGYMHWYQQKPGKLC:T5 952.5350 LLIYDTSKLC:T6 786.4468 LASGVPSR

LC:T7 4483.0023FSGSGSGTEFTLTISSLQPDDFATYY CFQGSGYPFTFGGGTK

Carbamidomethyl C

LC:T8 488.3079 VEIKLC:T10 1946.0270 TVAAPSVFIFPPSDEQLKLC:T11 1797.8952 SGTASVVCLLNNFYPR Carbamidomethyl CLC:T13 560.3191 VQWKLC:T14 2135.9687 VDNALQSGNSQESVTEQDSKLC:T15 1502.7585 DSTYSLSSTLTLSKLC:T18 1875.9269 VYACEVTHQGLSSPVTK Carbamidomethyl CLC:T19 523.2623 SFNRLC:T34 1286.6739 EPQVYTLPPSR

*Modification sites are marked in bold in peptide sequence.

4mAb Tryptic Digestion Standard

[ CARE AND USE MANUAL ]

V. EXAMPLE LC-UV, LC-MS CONDITIONS AND REPRESENTATIVE DATA

a. Peptide mapping with formic acid, difluoroacetic acid, and trifluoroacetic acid mobile-phase conditions

Gradient:

Time Flow rate (min) (mL/min) %A %B Curve

0.00 0.2 99.5 0.5 6 10.00 0.2 99.5 0.5 6 75.00 0.2 60.0 40.0 6 76.00 0.2 20.0 80.0 6 80.00 0.2 20.0 80.0 6 81.00 0.2 99.5 0.5 6 100.00 0.2 99.5 0.5 6

MS settings:MS instrument: Xevo™ G2-XS QTof

Mode: Full scan with fragmentation mode

Mass range: 100–2000 m/z

Cone voltage: 50 V

Capillary voltage: 3.5 KV

Desolvation temp.: 500 °C

Fragmentation: 20–40 V

Figure 2. Representative UV chromatograms of the mAb Tryptic Digestion Standard as obtained using either 0.1% formic acid (top), DFA (middle) or TFA (bottom). Chromatograms were acquired with an ACQUITY UPLC H-Class Bio System and an ACQUITY UPLC Peptide CSH C18 Column.

0

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25

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100

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100

0

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75

100

6562.56057.55552.55047.54542.54037.53532.53027.52522.52017.51512.510 67.5 70 min

0.1% FAPc*=298

0.1% DFAPc*=420

0.1% TFAPc*=469

LC instrument: ACQUITY™ UPLC™ H-Class Bio

Column: ACQUITY UPLC Peptide CSH C18, 130 Å, 1.7 µm, 2.1 x 150 mm (p/n: 186006938)

Temp.: 80 °C

Mobile phase A: 0.1% Formic acid (v/v), difluoroacetic acid (DFA) or trifluoroacetic acid (TFA) in water

Mobile phase B: 0.1% Formic acid (v/v), difluoroacetic acid (DFA) or trifluoroacetic acid (TFA) in acetonitrile

Flow rate: 0.2 mL/min

Sample temp.: 10 °C

UV detection: 214 nm

Seal wash: 90% Water/10% acetonitrile (v/v)

Needle wash: 90% Water/10% acetonitrile (v/v)

Reconstitution: 80 µL 0.1% formic acid in water

Injection volume: 10 µL

http://www.waters.com/waters/partDetail.htm?partNumber=186006938

5mAb Tryptic Digestion Standard

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Figure 3. BPI chromatogram and peak assignments for mAb Tryptic Digestion Standard. This chromatogram was acquired with a Xevo G2-XS QTof Mass Spectrometer using 0.1% TFA and an ACQUITY UPLC Peptide CSH C18 Column. Data processing was performed with UNIFI™ v1.8.

Figure 4. Processed TIC chromatogram of the mAb Tryptic Digestion Standard acquired with the BioAccord System using 0.1% formic acid and an ACQUITY UPLC BEH C18 Column.

100000

0

2e5

BP

I [C

ount

s]

3e5

4e5

1510 20 25 30Retention time

35 40 45 50 55 60 min

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1e7TIC

[Cou

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2e7

3e7

5 10 15 20 25 30Retention time

35 40 45 50 55 60 min

TIC (m/z 50-2000) after automated data

b. Routine peptide mapping with the BioAccord System

MS Setting:MS instrument: ACQUITY RDa™ Mass Detector

Mode: ESI positive, full scan with fragmentation mode

Mass range: 50–2000 m/z

Cone voltage: 30 V

Capillary voltage: 1.2 KV

Desolvation temp.: 350 °C

Fragmentation: 60–120 V

LC Instrument: ACQUITY UPLC I-Class PLUS

Column: ACQUITY UPLC BEH C18, 130 Å, 1.7 µm, 2.1 x 100 mm (p/n: 186002352)

Temp.: 65 °C

Mobile phase A: 0.1% Formic acid (v/v) in water

Mobile phase B: 0.1% Formic acid (v/v) in acetonitrile

Flow rate: 0.25 mL/min

Sample temp.: 6 °C

Reconstitution: 200 µL 0.1% formic acid in 18.2 MΩ water

Injection volume: 5 µL

Gradient: 1%–40% 0.1% formic acid in acetoni-trile (B) over 65 min

http://www.waters.com/waters/partDetail.htm?partNumber=186002352

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Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

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Waters, The Science of What’s Possible, BioAccord, ACQUITY, UPLC, Xevo, UNIFI, BioResolve, and RDa are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2019 Waters Corporation. Produced in the U.S.A. February 2019 720006359EN IH-PDF

Figure 5. Mass spectrum and fragmentation view of the heavy chain T15 peptide (MW = 6713.3144 Da). The monoisotopic peak of T15 peptide was observed in the raw spectrum, and the sequence of T15 was verified by a fragmentation spectrum containing 64 matching fragment ions.

T:15 raw spectrum T:15 peptide fragment ions

0

2.5e5

Observed mass [m/z]

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7.5e5

1e6

1.25e6

1.5e6

1.75e6

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2.25e6

Inte

nsity

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nts]

1343 1343.5 1344 1344.5 1345 1345.5 1346 1346.5

VI. CAUTIONARY NOTEDepending on the user’s application, these products may be classified as hazardous following their use and as such are intended to be used by professional laboratory personnel trained in the competent handling of such materials.

Responsibility for the safe use and disposal of products rests entirely with the purchaser and user. This product is research only and not for IVD use. The Safety Data Sheet (SDS) for this product is available at www.waters.com/sds.

VII. ORDERING INFORMATIONProduct description Part numbermAb Tryptic Digestion Standard 186009126Humanized mAb Mass Check Standard 186009125mAb Subunit Standard 186008927ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 µm, 2.1 x 100 mm, 1/pkg 186002352ACQUITY UPLC Peptide BEH C18 Column, 300 Å 1.7 µm, 2.1 x 150 mm, 1/pkg 186003687

ACQUITY UPLC Peptide CSH C18 Column, 130 Å 1.7 µm, 2.1 x 150 mm, 1/pkg 186006938

ACQUITY UPLC Peptide HSS T3 Column, 100 Å 1.8 µm, 2.1 x 150 mm, 1/pkg 186008756ACQUITY UPLC Protein BEH C4 Column, 300 Å 1.7 µm, 2.1 x 50 mm, 1/pkg 186004495BioResolve RP mAb Polyphenyl Column, 450 Å 2.7 µm, 2.1 x 50 mm, 1/pkg 186008944

http://www.waters.comhttp://www.waters.com/waters/en_GB/Safety-Data-Sheets/nav.htm?cid=134855469&alias=Alias_SDS_CORPORATEhttp://www.waters.com/waters/partDetail.htm?partNumber=186009126http://www.waters.com/waters/partDetail.htm?partNumber=186009125http://www.waters.com/waters/partDetail.htm?partNumber=186008927http://www.waters.com/waters/partDetail.htm?partNumber=186002352http://www.waters.com/waters/partDetail.htm?partNumber=186003687http://www.waters.com/waters/partDetail.htm?partNumber=186006938http://www.waters.com/waters/partDetail.htm?partNumber=186008756http://www.waters.com/waters/partDetail.htm?partNumber=186004495http://www.waters.com/waters/partDetail.htm?partNumber=186004494