Routine Characterization of mabs and Other Proteins Patrick Boyce Biopharmaceutical Marketing Manager Europe and India 2011 Waters Corporation 1
Agenda Why? What scientific challenges? Technology Example application Sequence confirmation Identification and quantification of glycans & other PTMs 2011 Waters Corporation 2
Why the need to confirm sequence, identify PTMs and quantify PTMs? Regulatory guidelines such as ICH Q6B, and EMEA/CHMP/BWP/49348/2005 make it clear that t the primary structure should be confirmed, heterogeneity defined, product related substances characterized, and consistency demonstrated. 2011 Waters Corporation 3
Scientific challenges Confirming primary structure LC/MS of peptide map complicated by sub-stoichiometric modifications, and missed cleavages, which h may result in a very large quantity of peptides. Detecting, locating & quantifying modifications Qualitative & quantitative analysis of low mass modifications to high molecular mass samples Dynamic range challenge Specific MS challenges Multiple charge states, isotope patterns, co-eluting peptides, insource fragmentation, data interpretation Isobaric peptides p in peptide p map Detecting the monisotopic ions becomes more demanding as mass increases. 2011 Waters Corporation 4
Overview of Waters Technology for Biopharmaceutical Analysis ACQUITY UPLC H-Class Bio UV and fluorescence detectors Xevo G2 QTof MS BiopharmaLynx 1.2 Column chemistries AccQTag Ultra BEH C18 BEH130 & BEH300 C18 PST BEH300 C4 PrST columns BEH Glycan columns BEH200 SEC columns Protein-Pak TM Hi Res IEX columns MassPREP Micro Desalting column 2011 Waters Corporation 5
ACQUITY UPLC H-Class Bio Ultimate chromatographic performance Low dispersion system sub-2 µm columns Biocompatible system Eliminate system corrosion Best sample recovery Flexibility, robustness Suitable for RP, IEX, SEC, HILIC Robustness tested for high salt AutoBlend+ for dial-up buffers Needle in flow path design Low carryover performance 2011 Waters Corporation 6
Mass accuracy, resolution and dynamic range Accurate mass of monoisotopic peptide for identification QuanTof technology: Improved quantitation Enables identification of larger peptides Disulfide-linked peptides Missed Cleavage Digests: AspN, LysC, GluC TOF >20,000 RESOLUTION QuanTof TM Low ppm MMA over >10 4 Dynamic Range 2011 Waters Corporation 7
Xevo G2 Technical Note Peptide Mass Monoisotopic peak (% of total) 1 Relative Monoisotopic Peak Height 2 Isotopic Envelope Dynamic Range 3 2,000 31 93 1.1 5,000 5.7 26 3.8 10,000 0.3 2.1 47.6 15,000 0.018 0.14 714.2 20,000 0.001 0.01 10000 ~9:1 ~9:1 The largest peptide in most antibody tryptic peptide maps: HC T15 (DYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTK). Mass 6712.3071 Da. Dynamic Range Required ~1400 2011 Waters Corporation 8
UPLC/MS E to Comprehensively Catalog Complex Samples UPLC/MS E comprehensively catalogs complex samples in a single analysis. It delivers high quality, unambiguous exact mass precursor and fragment ion data quickly and easily 2010 Waters Corporation
MS TIME MS E 2011 Waters Corporation 10
What BiopharmaLynx Does Automates data processing Facilitates comparisons between a reference standard and batches of experimental samples. Useful for: demonstrating consistency of batch with material used for clinical studies demonstrating comparability of reference product with potential new biosimilar 2011 Waters Corporation 11
Application - Introduction and Context Company X is an international company with a powerful track record in small molecules. They wish to expand their product range and plan to make a biosimilar of an antibody They sent us two samples The innovator product Their copy of that product They wished to verify that they had successfully mimicked the innovator product. http://en.wikipedia.org/wiki /File:Antibody2.JPG 2011 Waters Corporation 12
UPLC/MS of Intact Control and Check Samples With Data Processed by BiopharmaLynx 1.2 2011 Waters Corporation 13
Intact Protein Analysis Workflow Sample Preparation + + UPLC Xevo QTof MS Bioinformatics 2011 Waters Corporation 14
Control and Check Antibodies Processed automatically by BiopharmaLynx 1.2 CONTROL Consistent 64 Da difference in antibody masses for each form CHECK 2011 Waters Corporation 15
High Resolution Separation of a Reduced Monoclonal Antibody Mass Spectrum LC Mass Spectrum HC LC Chromatogram HC Waters BEH300 C4 1.7 µm 2.1 x 50 mm A: 0.1% Formic in Water B: 0.1% 01% Formic in ACN 25-35 %B over 15 min 2011 Waters Corporation 16
Automatically Deconvoluted Spectrum of the Light Chains in BiopharmaLynx The average masses of the Light Chains of the antibodies are identical and it is reasonable to suggest that they share the same chemical composition CONTROL CHECK Light Chains are identical 2011 Waters Corporation 17
Automatically Deconvoluted Spectrum of the Heavy Chains in BiopharmaLynx CONTROL CHECK The difference between the CONTROL and CHECK sample is -32.3 Da per heavy chain. This corresponds to approximately -64.6 Da in the intact antibody of the CHECK sample when compared with the CONTROL antibody. 2011 Waters Corporation 18
Glycan Characterization ACQUITY UPLC H-Class Bio with FLR detection BEH Glycan Column Released glycans, gy 2-AB Labelled 2011 Waters Corporation 19
HILIC-FLR-MS analysis of 2-AB labeled released glycans gy Biosimilar glycans were qualitatively consistent with the innovator mab therefore differences are not due to glycoforms 2011 Waters Corporation 20
Confirmation of Sequence By UPLC-MS E of Trypsin Digest of Control and Check Samples With Data Processed by BiopharmaLynx 1.2 2011 Waters Corporation 21
Company X Samples and Analysis for Peptide Map 2 samples CONTROL (reference) and CHECK (copy) RapiGest-assisted 4-hour tryptic digestion ACQUITY UPLC separation (BEH130 C18 column) MS E detection for simultaneous qualitative & quantitative analysis Data Interpretation performed by BiopharmaLynx CONTROL mab Light Chain (LC) and Heavy Chain (HC) sequences loaded 2011 Waters Corporation 22
Chromatograms of Control and Check Samples CONTROL CHECK 2011 Waters Corporation 23
Sequence Coverage Light Chain Coverage Map for Light Chains for CONTROL and CHECK samples is identical (100%) 2011 Waters Corporation 24
Mirror View of HT34-35s in Biopharmalynx Processed MS trace HT34-35 in CONTROL sample Missing peptide in CHECK sample Unknown peak in CHECK sample 2011 Waters Corporation 25
Mirror View of HT35s in Biopharmalynx Processed data HT35 in Control Sample Missing peptide in CHECK sample Unknown peak in Check Sample 2011 Waters Corporation 26
MS E Spectra of Control mab HT35 (EEMTK) and HT34-35 (EPQVYTLPPSREEMTK) T35 (EEMTK) Control Sample Fragment Ion Data MS E collects all the exact mass precursor and fragment ion data for every peak T34 35 (EPQVYTLPPSREEMTK) Control Sample Fragment Ion Data 2011 Waters Corporation 27
MS E Spectra of Control mab HT35 (EEMTK) and HT34-35 (EPQVYTLPPSREEMTK) T35 (EEMTK) Control Sample Fragment Ion Data BiopharmaLynx automatically T34 35 (EPQVYTLPPSREEMTK) Control Sample Fragment interprets Ion Data the MS E data and provides sequence confirmation 2011 Waters Corporation 28
MS E Spectra of Check mab HT35 (DELTK) and HT34-35 (EPQVYTLPPSRDELTK) T35 (DELTK) Check Sample Sequence T34 35 35 (EPQVYTLPPSRDELTK) Check Sample confirmation of unknown peaks in CHECK sample 2011 Waters Corporation 29
Conclusions regarding g Data Primary amino acid sequence is different between CONTROL and CHECK 3-amino acid stretch where two amino acids are different and resultant mass difference is 32Da EEMTK vs DELTK Clone appears to have been manufactured using the sequence indicated in Drugbank which is different to the CONTROL mab A simple LCMS E peptide map saved much time and money in avoiding scale-up for that clone 2011 Waters Corporation 30
Simultaneously Confirming Sequence, Identifying PTMs and Quantifying PTMs with UPLC-MS E of Trypsin Digest of Control and Check Samples With Data Processed by BiopharmaLynx 1.2 2011 Waters Corporation 31
Identifying PTMs with BiopharmaLynx Scrambled disulfides 2011 Waters Corporation 32
Identifying PTMs with BiopharmaLynx List of available modifiers custom modifiers df can be added List of allowed fixed or variable modifiers 2011 Waters Corporation 33
Chromatograms of Control and Check Samples CONTROL CHECK 2011 Waters Corporation 34
Automated PTM identification & quantification of PTMs e.g. HT21 (DTLMSIR) CONTROL: 9% M-oxidized HT21 CHECK: 18% M-oxidized HT21 Unmodified HT21 2011 Waters Corporation 35
Summary Routine Characterization UPLC/MS on intact and reduced proteins UPLC with FLR detection on released glycans UPLC/ MS E analysis of peptide map o Separation of peptide map on ACQUITY UPLC o MS E data acquisition mode to get the exact mass precursor and fragment ion data on every detectable peak across an entire chromatogram with a single analysis Automated t data processing with BiopharmaLynx 2011 Waters Corporation 36
Search www.waters.com for: Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced LC and MS techniques Development of Mass Spectrometric and Informatics Workflows for the Automated Assessment of Biosimilarity for a Candidate Biosimilar Antibody Fast and Automatic Mapping of Disulfide Bonds in a Monoclonal Antibody using SYNAPT G2 HDMS and BiopharmaLynx 1.3 Comprehensive and Routine Characterization of Proteins and Peptides using an Integrated Waters LC/MS Workflow High Sequence Coverage Peptide Mapping of a Monoclonal Antibody with UPLC/MS E Application Solutions for Biopharmaceuticals: A Focus on Protein Therapeutics RapiGest SF ACQUITY UPLC H-Class Bio BiopharmaLynx Xevo G2 QTof 2011 Waters Corporation 37
Acknowledgements Scott Berger Asish Chakroborty Weibin Chen St John Skilton 2011 Waters Corporation 38