Critical Quality Attribute Assessment by Peptide Mapping Using LC/MS with Superficially Porous Columns

Transcription

1 Critical Quality Attribute Assessment by Peptide Mapping Using LC/MS with Superficially Porous Columns

2 Analytical groups are tasked with method development, sample analysis, and method transfer DNA Vector Establishment of genetically engineered cells (e.g. CHO) that produce desired product Biomarker ID & validation Samples Human gene HT Clone mab Generation Platform Screening & Cell Culture Scale-up & Production e.g. transgenic mice Selection Samples Samples Samples QA/QC Methods Analytical Groups Develop analytical methods; analyze samples from other functions, methods transfer Centrifuge Samples Samples Market Manufacturing Clinical Pre-clinical Chromatography Column #3 Chromatography Column #2 Chromatography Column # 2

3 Critical Quality Attributes & Testing Methods Not just used for monoclonal antibodies same techniques are used for any potentially therapeutic protein RP Disulfide Shuffling SEC Aggregation Pyro- Glutamate IEC LC/MS Intact mass Fragment mass Identification PTM ID & locations Glycan ID Glycosylation (G, G, G2) HILIC Fragmentation (Hinge) Truncation (Lys,, 2) IEC RP Deamidation /Oxidation IEC 3

4 Peptide Mapping Workflow Solutions to Accelerate Biomolecule Characterization A(3-6) B(67-73) B(33-337) B(278-29) B ( ) Data Acquisition Peptide mapping chromatogram Peptide ID by MS/MS Sequence Matching Determine Post Translational Modifications Chromatographic Separation Reporting Sample Preparation Complete Agilent Workflow Solution Delivery of Results 4

5 Sample Preparation for Peptide Mapping A time consuming, labor-intensive process to do manually, with multiple steps that can be optimized. Sample Preparation Depletion of matrix proteins, enrichment of target protein, dialysis or desalting Reduce disulfide bonds, and block to prevent re-formation Denaturation, Reduction, & Alkylation Digestion Trypsin is the most common digestion enzyme, but others may be called for depending on the protein and experiment. Sample concentration, enrichment for specific PTMs, or desalting Enrichment & Cleanup 5

6 AssayMAP Bravo Sample Prep Automation Peptide mapping Detect 6

7 AssayMAP Sample Prep Portfolio Antibody Purification Immunoaffinity Purification Fractionation Protein Digestion Peptide Clean Up Protein Clean Up Phosphopeptide Enrichment N-glycan Sample Prep 7

8 General Considerations for Peptide Mapping Column Selection Instrument capabilities and requirements - UV vs MS detection or both - Pressure capabilities Mobile phase requirements - High ph required for sample - TFA vs Formic acid Sample - Hydrophilic vs hydrophobic peptides - Larger polypeptides present Column dimensions - Generally prefer longer columns, especially for more complicated maps - 5, 5, and 25 mm lengths available - Use 2. mm i.d. for MS-sensitivity - 3. and 4.6 mm i.d. also available - Smaller pore sizes ideal, usually -5 Å 8

9 Detectors for Peptide Mapping UV Often used in routine process measurements, QA/QC labs No peptide mass or structural information; relies on reproducibility of RT, peak area, and peak area ratios Traditionally uses TFA modifier MS Often used in discovery/early development stage, R&D labs MW and structural information (MS/MS) to assist with co-elution challenges, verify sequence coverage, and to ID unknown peaks when they appear Generally prefer formic acid modifier to avoid ion suppression from TFA 9

10 Mobile Phases for Peptide Mapping Ion pairing agents used for RP LC Increase retention of small, poorly retained peptides Block exposed silanol sites on silica-based columns Acid helps solubilize and ionize peptide and protein samples TFA works well on traditional silicabased columns, but formic acid does not Formic acid produces broad, asymmetric peaks Poor resolution and poor peak capacity Need column solution for formic acid modifier AdvanceBio Peptide Plus Charged surface chemistry solves the problems associated with formic acid

11 Agilent Bio-LC Column Portfolio Agilent Bio-LC Columns Affinity Reversed Phase HILIC Size Exclusion Ion Exchange Bio-Monolith Protein A AdvanceBio Peptide Plus AdvanceBio Glycan Mapping AdvanceBio SEC Bio mab Bio-Monolith Protein G Multiple Affinity Removal System AdvanceBio Peptide Mapping AdvanceBio RP mab AdvanceBio Oligonucleotides AdvanceBio Desalting-RP ZORBAX RRHD 3-HILIC Bio SEC-3 Bio SEC-5 ProSEC 3S ZORBAX GF-25 Bio IEX (SAX, SCX, WAX, WCX) PL SAX PL SCX Bio-Monolith (QA, DEAE, SO 3 ) PLRP-S ZORBAX GF-45 ZORBAX RRHD 3Å,.8 μm AdvanceBio Amino Acid Analysis ZORBAX Amino Acid Analysis ZORBAX 3SB Poroshell 3

12 Agilent Bio-LC Column Portfolio Agilent Bio-LC Reverse Phase Columns for Protein Analysis Intact Proteins (& Fragments) Peptides Amino Acids PLRP-S, 5 µm, Å AdvanceBio Peptide Plus AdvanceBio Amino Acid Analysis AdvanceBio RP mab AdvanceBio Peptide Mapping ZORBAX Amino Acid Analysis ZORBAX RRHD.8 µm, 3 Å ZORBAX Eclipse Plus C8 ZORBAX 3SB ZORBAX 3SB* Poroshell 3 AdvanceBio Desalting-RP *unusually large pore size for peptide mapping, but some people really like it nonetheless 2

13 AdvanceBio Peptide Plus Poroshell particle UHPLC performance at lower pressure Unique charge hybrid/c8 bonded phase Improved peak shape and peak capacity for peptide analysis with formic acid and MS detection To provide AdvanceBio Peptide Plus that gives benefits in accuracy and reproducibility of results for characterization of mab identity, PTMs 3

14 Columns Individually Tested for Efficiency AdvanceBio Peptide Plus Column 4

15 Column Lots QA Tested Specifically for Peptide Separations Peptides Standard (p/n G ) AdvanceBio Peptide Plus Column 5

16 LC/MS Instrumentation 29 Infinity II LC system 6545XT AdvanceBio LC/Q-TOF BioConfirm B.8 for intact protein and peptide mapping data analysis 6

17 6545XT Features for Peptide Mapping Sensitive peptide detection featuring Agilent Jet Stream Quick-start peptide mapping method Access low intensity peptides/ptms with the new IterativeMS/MS mode Sub-ppm mass accuracy with 5k resolution from improved beam optics Peptide fragmentation performance verification at install Ease of maintenance with vent-free capillary removal

18 Method Conditions Parameter 29 Infinity II LC Column AdvanceBio Peptide Plus 2. 5mm, 2.7µm, 2Å Mobile phase A:.%FA B:.% FA in ACN Time (min) %B 3 3 Gradient Post time min Inj vol µl Sample thermostat 5 C Post time 5 min Column temp 55 C Flow rate.5 ml/min Sample trastuzumab digest (ug/ul) All results shown are with formic acid mobile phase Parameter 6545XT AdvanceBio LC/Q-TOF Ion mode Positive ion mode, dual AJS ESI (profile) Drying gas temp 325 C Drying gas flow 3 L/min Sheath gas temp 275 C Sheath gas flow 2 L/min Nebulizer Capillary voltage Fragmentor voltage Skimmer voltage Oct RF Vpp Acquisition parameters 35 psi 4 V 75 V 65 V 75 V Data were acquired at 2 GHz Extended Dynamic Range MS mass range 7 m/z MS/MS mass range 5 7 MS scan rate (spectra/second): 8 MS/MS scan rate (spectra/second): 3 Ramped collision energy Charge state Slope Offset and > Data analysis BioConfirm software B.8.8 8

19 Effect of Flow Rate on Peak Capacity AdvanceBio Peptide Plus Column x ml/min Increasing the flow rate from. ml/min to.5 ml/min leads to an increase of twice the peak capacity x 8.2 ml/min.5 x 8.3 ml/min.5 x 8.4 ml/min.5 x ml/min.5 x ml/min.5 TIC Counts vs. Acquisition Time (min) ug trastuzumab digest 9

20 Effect of Gradient Length on Peak Capacity AdvanceBio Peptide Plus Column x x 8 min Increasing the gradient time from min to 6 min leads to an increase 2.5 times peak capacity, After 3min, no significant increase in peak capacity 2 min.5 x min.4 x min.4 x min.4 x min.4 TIC Counts vs. Acquisition Time (min) ug trastuzumab digest 2

21 Effect of Gradient Slope on Peak Capacity AdvanceBio Peptide Plus Column x8.5-4% in 3min Slope.3%/min Higher efficiency sharper peaks Increased sensitivity Peak capacity = 42 Sequence coverage = 96.69% x % in 6min Slope.6%/min Improved resolution Peak capacity = 59 Sequence coverage = 94.59%.5 TIC Acquisition Time (min) ug trastuzumab digest 2

22 Effect of Temperature on Peak Capacity AdvanceBio Peptide Plus Column x6 Higher temperature produces narrower peaks, improving resolution Change in temperature changes selectivity ~2% more peaks resolved by increasing the column temperature to 55 C 2 45 o C Pc = 54 x o C Pc = 6 LC/MS Chromatogram Co unts vs. Acquisition Time (min) ug trastuzumab digest 22

23 Back Pressure Characterization AdvanceBio Peptide Plus Column 2. x 5 mm 6 bar pressure maximum Temperature *pressure reading observed (max) Elevated temp produces narrower peak bands and lower pressure 23

24 Good Peak Shape & Retention Time Stability with High Mass Load AdvanceBio Peptide Plus Competitor Sample: 3-peptide mixture in.%fa Bradykinin Angiotensin II Venin substrate Competitor 2 Competitor 3 Key for transferring methods between LC/MS and LC/UV All 2. x 5 mm columns 24

25 Highly Reproducible Separations Co unts x Hydrophilic Hydrophobic AdvanceBio Peptide Plus Column Acquisition Time (min) n = %RSD Peak no. RT FWHM Area Height Consistent chromatography RSD values demonstrate the robustness of the method and precision of the LC system Especially crucial for LC/UV peptide mapping 25

26 Lifetime Testing Retention time % Column: AdvanceBio Peptide Plus Sample: peptide standard (59-583) Injection: 3 µl Flow rate:.5 ml/min Mobile phase: A.%FA B.% FA in ACN R t dropped < % Plates count % The column has subjected to injections and lost no more than % of the initial plate and retention value at the end of the test % Plate count Plates dropped < % Good chemical stability under formic acid conditions 26

27 Column Options for Alternate Selectivity x AdvanceBio Peptide Mapping Peptide Standard (p/n ) x AdvanceBio Peptide Plus FWHM Better C ounts v s. Acquisition Time (min).4.2 AdvBio Peptide Mapping AdvBio Peptide Plus TIC. Bradykinin frag Bradykinin 3. Angiotensin II (human) 4. Neurotensin 5. Angiotensin I (human) 6. Renin substrate porcine 7. [Ace-F-3,-2 H-] Angiotensinogen (-4) 8. Ser/Thr Protein Phosphatase (5-3) 9. [F4] Ser/Thr Protein Phosphatase (5-3). Melittin (honey bee venom) Peptide peak 27

28 mab Peptide Mapping x8 5 LC/MS of mab tryptic digest on 2. x 5mm AdvanceBio Peptide Plus Column Hydrophilic Hydrophobic 4 3 Counts Acquisition Time (min) Hydrophilic peptide retention Narrow Peaks with baseline resolution Hydrophobic peptide elution Reduced and fast analysis time Critical and desired peptide mapping components to achieve fast, selective, and highly efficient peptide separations across a wide dynamic range. 28

29 Tryptic Peptides of Trastuzumab 29

30 LC/MS Peptide Map AdvanceBio Peptide Plus Column sequence coverage 99.25% mass accuracy of 5 ppm 3

31 MassHunter BioConfirm for Peptide Mapping Sample Prep Separation Detection Data Processing & Report

32 Counts Separation of Oxidized Peptides x DLTMISR peptide Unmodified peptide x x DLTMISR Unmodified peptide b -H2O L,I y b y b y T R y 2-NH y y b 4-H2O Counts v s. Mass-to-Charge (m/z) DLTMISR Oxidized peptide AdvanceBio Peptide Plus Column MS/MS spectra y Oxidized peptide L,I y y b y b -H2O Acquisition Time (min) y -NH b -H2O EIC Relative % of Oxidation =.69% Counts vs. Mass-to-Charge (m/ z) Increase in mass (6Da) 32

33 Separation of Deamidated Peptides NTAYLQMNSLR Deamidated peptides NTAYLQMNSLR peptide Relative % of Deamidation =.2% 5.9% EIC AdvanceBio Peptide Plus Column ASQDVNTAVAWYQQKPGK Non-deamidated peptide Relative % deamidation = 9.% Deamidated peptide ASQDVNTAVAWYQQKPGK peptide Relative % of Deamidation = 9.% 33 non-deamidated peptide NTAYLQMNSLR NTAYLQMNSLR 5.9% NTAYLQMNSLR.23% NTAYLQMNSLR 5.5% NTAYLQMNSLR.97% NTAYLQMNSLR.24% NTAYLQMNSLR 2.5% NTAYLQMNSLR.2%

34 Critical Quality Attributes & Testing Methods Not just used for monoclonal antibodies same techniques are used for any potentially therapeutic protein RP Disulfide Shuffling SEC Aggregation Pyro- Glutamate IEC LC/MS Intact mass Fragment mass Identification PTM ID & locations Glycan ID Glycosylation (G, G, G2) HILIC Fragmentation (Hinge) RP Deamidation /Oxidation IEC With a Multi-Attribute Method, peptide mapping can monitor many of these CQAs simultaneously. Truncation (Lys,, 2) IEC 34

35 Multi-Attribute Method (MAM) 35

36 Separation of Deamidated Peptides VVSVLTVLHQDWLnGK or VVSVLTVLHqDWLNGK m/z , doubly charged peptide Traditional silica columns don t resolve deamidation as well as charged surface columns. Traditional Silica Column AdvanceBio Peptide Plus Determination of deamidated peptides is an important goal of peptide mapping. Chromatographic resolution is important. MS/MS confirms which peak corresponds to which deamidation site. AdvanceBio Peptide Plus enhances analysis because it has higher selectivity for deamidated forms of peptide over the normal form.

37 Choosing a Column Chemistry For LC/MS work, AdvanceBio Peptide Plus with formic acid is the first choice. Best peak shape with formic acid, high peak capacity, high mass load tolerance, excellent PTM resolution (especially deamidation), peak shape and recovery of hydrophobic peptides, unique selectivity Exception : Sample is especially rich in small, hydrophilic peptides. Poor retention of small, hydrophilic peptides is a feature common to charged surface columns. In this case, try AdvanceBio Peptide Mapping or Eclipse Plus C8 first. Exception 2: Different selectivity is needed to separate a key pair. Charged surface chemistry AdvanceBio Peptide Plus vs Regular silica chemistry AdvanceBio Peptide Mapping Eclipse Plus RRHD ZORBAX 3 37

38 Choosing a Column Chemistry Sample Detection First Choice of column Alternative column Peptides LC/MS (FA) AdvanceBio Peptide Plus AdvanceBio Peptide Mapping LC/UV AdvanceBio Peptide Mapping AdvanceBio Peptide Plus ZORABX RRHD 3SB-C8 or Diphenyl For maximum flexibility to transfer methods across instrument platforms, use AdvanceBio Peptide Plus with formic acid. 38

39 Method Development Tips & Reminders Bonded Phase: C8 is routinely used. Try AdvanceBio Peptide Plus first; for alternate selectivity or highly hydrophilic samples try AdvanceBio Peptide Mapping or a ZORBAX column. Column dimension: Good start with 2.mm 5mm, for higher resolution use longer column Gradient: Acetonitrile : Water with.% formic acid, other organic solvent substitutions can be used for different selectivity Starting gradient time: -4% in 3min (.3%/min) Temperature: Higher column temperature can dramatically improve both resolution and recovery (6 C max) Desalt peptide mixture prior to injection Sample handling may cause artifacts (peptide modifications) 39

40 Troubleshooting Reasons for < % Sequence Coverage Question to ask: Looking at the protein sequence, what s missing? Very small and/or very hydrophilic peptides? If using AdvanceBio Peptide Plus, try AdvanceBio Peptide Mapping for better retention and/or resolution. Very large and/or very hydrophobic peptides? Can you go to a higher percent organic mobile phase? Are peptides sticking to sample tubes or otherwise lost during sample preparation? Or on the LC? If using AdvanceBio Peptide Mapping, try AdvanceBio Peptide Plus - May even need to consider a C8 column rather than C8 Very large, or very small peptides of any hydrophobicity? Consider a different digestion enzyme 4

41 More Information How-to Guide Application Notes: LC/MS Analysis of Peptide Mapping with Formic Acid Ion-Pairing Agent ( EN) Enhancing the Quality of Peptide Mapping Separation for the Analysis of Post- Translational Modifications ( EN) 4

42 Standards & Reagents Peptide Standard (p/n ) Co unts x Blank A cquisition Time (min) 7 Trypsin Digest Methylated BSA Standard (p/n G99-85) 9 8. Bradykinin frag Bradykinin 3. Angiotensin II (human) 4. Neurotensin 5. Angiotensin I (human) 6. Renin substrate porcine 7. [Ace-F-3,-2 H-] Angiotensinogen (-4) 8. Ser/Thr Protein Phosphatase (5-3) 9. [F4] Ser/Thr Protein Phosphatase (5-3). Melittin (honey bee venom) HSA peptides Standard (p/n G ) Trypsin. AAFTE[CAM][CAM]QAADK 2. YLYEIAR 3. LVNEVTEFAK 4. KVPQVSTPTLVEVSR 5. RP[CAM]FSALEVDETYVPK 6. AVMDDFAAFVEK 7. HPYFYAPELLFFAK 42

43 Summary High efficiency, high resolution separation of peptides using AdvanceBio Peptide Plus column MS-compatible AdvanceBio Peptide Plus column delivers high peak capacity with sharp and narrow peaks using MS-friendly formic acid modifier mobile phase conditions. AdvanceBio Peptide Plus and AdvanceBio Peptide Mapping are superficially porous columns that yield low back pressures & STM-like performance, allowing peptide mapping separations to be run on both HPLC and UHPLC systems Well-resolved PTM modified peptide peaks enabled reliable mab peptide maps and development of Multi-Attribute Methods High quality separations in ~3 minutes with AdvanceBio Peptide Plus make peptide mapping more time-effective Multiple column options for every customer s unique needs 43