LC/MS Based Quantitation of Intact Proteins for Bioanalytical Applications
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1 LC/MS Based Quantitation of Intact Proteins for Bioanalytical Applications Alex Zhu, Ph.D. Agilent Technologies Wilmington, DE ASMS,
2 Outline Introduction on intact protein quantitation Agilent Solutions for intact protein quantitation (standard flow UHPLC/6545XT) Quantitation of intact mab in neat solution Quantitation of intact mab in rat plasma Quantitation of antibody drug conjugate in rat serum 2
3 Current Techniques for Protein Quantitation in Biological Matrices Ligand-binding assays (LBAs) - High sensitivity; high throughput - Require suitable capture and detection reagents; less specific, can be affected by the presence of anti-drug antibodies in the samples MS based - High specificity; wide dynamic range; faster method development; quantify multiple proteins simultaneously; quantify PTMs, degradation products, metabolites simultaneously 3
4 MS Based Protein Quantitation Strategies MS based Peptide approach - Digestion is time consuming resulting in limited throughput - Digestion efficiency and reproducibility requires extensive evaluation - Selection of surrogate peptide is not simple - Optimization of MRM method requires a lot of work - Loss of essential information of the intact protein can lead to ambiguous quantification Intact approach - Higher throughput - Specificity allows differentiation between closely related proteoforms - Offering unique possibility to quantify therapeutic or clinically relevant metabolites as well as postdose or pose-translational modified proteins - More accurate quantitation 4
5 Challenges for Intact mab Quantitation Using LC/MS Challenges Causes Possible Solutions Sensitivity Specificity Chromatography Data Analysis Diluted peak intensity due to formation of multiple charge states Interference from endogenous IgGs Limited separation efficiency and bad peak shape due to heterogeneity Single charge state? Summation of several charge states? Deconvoluted Spectrum? More sensitive instruments; ways to reduce number of charge states Sample preparation techniques; middle-down approaches; Higher res instr. (>600k) with fast enough acquisition rate for quant Better column; Alternative separation mechanisms (HILIC, etc.) Need evaluation 5
6 Example: J&J (Jun, 2016) J&J Sample Affinity Purification Flow rate Intact mab in Plasma (Sigma SiluLiteMab and SiluMab, SiluMab is heavy labeled and used as IS) Magnetic Beads Standard flow, 0.4 ml/min Mass Spec TripleTOF API 5600 Quant analysis Using height of deconvoluted spectra LLOQ Spectrum at LLOQ level Linear range 60 ng on-column (1 µg/ml with 60 µl inj) Yes (large mass difference >100ppm at all levels) 1 order (due to limit of binding capacity on beads) 6
7 Example: Novartis (Jan 2017) Novartis Article Sample Intact InfliximAb (Remicade) in rat serum Affinity LB-MSIA (Ligand Binding-Mass Purification Spectrometric Immunoassay) Flow rate 0.2 ml/min Mass Spec QE Quant analysis LLOQ Spectrum at LLOQ level Linear range Using sum of EICs 6 ng on-column Yes 2 orders ( µg/ml with 60 µl injection) 7
8 6545XT Features for Large Biomolecule Analysis Excellent protein spectral clarity from ultra-low TOF vacuum (10E-8) One-click optimization for large molecules with SWARM autotune Capable of analyzing very large molecules, with a variable mass range up to 30k m/z Ease of maintenance with vent-free capillary removal Protein performance verification at install, and includes quick-start protein method 8
9 Intact Protein Raw Spectral Quality Glycosylated Intact Excellent raw spectral quality to detect and identify minor isoforms such as loss of amino acids, minor glycoforms, and other PTMs. Intact NIST mab Analysis (0.5 µg injection) 9
10 Intact Protein Deconvoluted Mass Accuracy G0F + G1F 1.44 ppm G1F + G1F ppm As seen in the MaxEnt raw spectra, the increased spectral Deconvolution clarity leads to improved deconvolved results, both in accuracy and detection of minor isoforms. G0F + G0F ppm G1F + G2F 0.78 ppm Maximum Entropy deconvolution preserves low intensity signals for investigation of heterogeneity. (G0F+G0F) - GlcNAc (G0F+G1F) - GlcNAc (G1F+G1F) - GlcNAc (G1F+G2F) - GlcNAc (G2F+G2F) - GlcNAc G2F + G2F ppm 3.54 ppm Intact NIST mab Analysis (0.5 µg injection) 6/14/
11 How to Obtain Good Intact Protein (mab) Spectra? Issues/complaints: - Bad peak to hump - Broad spectral peaks - Loss of resolution of distinct glyco-forms - Bad mass accuracy - Cleaning optics, changing parts don t help - What s Wrong?? 11
12 Key: Cleanliness of the LC!!! Check Your LC Carefully Before Blaming the MS Contaminants in the LC may not ionize, so won t be seen directly in the MS spectra, but could form complexes (adducts) with the proteins, causing the issues! - Contaminated bottles - Buffer (water & organic) - Tubing - Column - Valves - Fittings/capillaries 12
13 One Example: Contaminants from Newly Opened HPLC Grade Bottled Water Using Milli Q Water Using HPLC Grade Bottled Water 13
14 Quantitation of Intact Herceptin In Neat Solution ASMS Poster: WP
15 Chromatography Optimization Issue: Broad chromatographic peak, tailing, making it difficult for accurate quant at low levels. From J&J Article From Agilent app note ( EN) Typical TIC for intact mab, showing board peak (>8s FWHM) and tailing. 15
16 Chromatography Optimization: Column Temperature (Herceptin) RT FWHM = 11s with significant tailing 80 C FWHM = 5.5s with some tailing 16
17 Chromatography Optimization (Herceptin) FWHM = 5.5s with some tailing Optimized Chromatography FWHM = 2.1s with no tailing 17
18 Sensitivity and Linear Range of Glycosylated Intact mab Trastuzumab EIC Sum Calibration Concentration (ng on-column) Accuracy (%, n=6) Cal. Conc. %RSD (n=6) Linear Range: > 3.2 orders (31.6 pg 50 ng or 200 amol 350 fmol) Standard Flow (0.4 ml/min) Zoom in 6/14/
19 Data from Trastuzumab Dilution Series ng 0.1 ng ng 1.0 ng x x x x x Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu) ng 3.16 ng 10 ng 31.6 ng 50 ng x x x x Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu) x Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu) 3.16 ng x10 5 6/14/
20 Relative abundances of major glyco-forms Relative Abundances of Different Glyco-forms Across the Linear Dynamic Range ( ng) Relative abundances of 6 major glyco-forms for Herceptin at different levels ( ng) G0F/G1F G0F/G0F G0F/G2F G1F/G2F G0/G0F G2F/G2F ng 31.6ng 10ng 3.16ng 1ng 0.316ng 0.1ng ng 20
21 Mass Error, ppm Mass Accuracy Reproducibility (Accuracy and Precision) Across the Linear Dynamic Range ( ng) Excellent mass accuracy and reproducibility across the linear range ng 31.6 ng 10 ng 3.16 ng 1 ng ng 0.1 ng ng G0F/G1F G0F/G0F G0F/G2F G1F/G2F G0/G0F G2F/G2F 21
22 Mass Accuracy Reproducibility (Accuracy and Precision) 100 Replicate Runs of 10 ng Injections For most abundant glyco-form G0F+G1F ( ): - Average mass error: ppm - Standard Deviation: 1.58 ppm - Mass error range (-5.05 to 2.09 ppm) 10 ng on-column 6/14/
23 Normalized Intensity Response Reproducibility 100 Replicate Runs of 10 ng Injections For 100 replicate runs, sum of EICs of most abundant 12 spectral peaks: - Standard deviation = 1.5% #40 Run 2.0s FWHM # of Runs 6/14/
24 Quantitation of Intact Herceptin in Rat Plasma Acknowledgement: Kevin Bateman and Lisa Varicek (Merck) for data acquisition and discussions 24
25 Experimental Flow 1290 Infinity II 6545XT AdvanceBio LC/Q-TOF Trastuzumab spiked Rat plasma Rat plasma (for serial dilutions) SA- W SA- W AssayMAP Bravo MassHunter Quant 25
26 Amount on Column Starting Concentration in Plasma (ug/ml) Initial Quantity in 30uL of plasma used (ng) AssayMAP Elution Concentration (ug/ml) Amount on column with 2uL injection (ng)
27 Calibration Curve EICs (Three at Each Level) 27
28 Calibration Curve 2.5E E+06 y = 33916x R² = Agilent 1.5E+06 Sample Intact Herceptin in rat plasma 1.0E+06 Affinity Purification AssayMAP 5.0E+05 Flow rate Standard flow, 0.5 ml/min 0.0E E E E+04 Mass Spec 6545XT AdvanceBio Q-TOF Quant analysis LLOQ Using sum of EICs 0.12 ng on-column Zoom of bottom 4 points 6.0E E+04 Linear range 2.7 orders 2.0E E
29 Quantitation of Intact ADC (T-DM1) in Rat Serum ASMS Poster MP132 29
30 Calibration Curve for Deglycosylated T-DM1 Agilent Sample Intact deglycosylated T-DM1 in rat serum Affinity Purification AssayMAP Flow rate Standard flow, 0.5 ml/min Mass Spec 6545XT AdvanceBio Q-TOF Quant analysis LLOQ Linear range Using peak areas from deconvoluted spectra 2 ng on-column for deglycosylated T-DM1 2 orders (2-200 ng on-column, assuming 100% recovery) 30
31 Example Spectra and DAR Calculation Deglycosylated T-DM1 2ng On-column 6.4 ng On-column 31
32 DAR Distribution at Different Levels 32
33 Summary Neat Herceptin Herceptin in Rat Plasma T-DM1 (ADC) in Rat Serum Sample Prep NA AssayMAP for affinity purification AssayMAP for affinity purification and deglycosylation Flow rate Standard flow, 0.5 ml/min Standard flow, 0.5 ml/min Standard flow, 0.5 ml/min Mass Spec 6545XT 6545XT 6545XT LLOQ 31.6 pg on-column (Herceptin) 120 pg on-column (Herceptin) 2 ng on-column (Deglycosylated T-DM1) Spectral fidelity Accurate glyco profile down to LLOQ at 31 pg on-column Accurate glyfo profile down to LLOQ at 120 pg on-column Accurate DAR calculation down to LLOQ at 2 ng on-column Linear range 3.2 orders ( ng oncolumn, Herceptin) 2.7 orders (120 pg 60 ng oncolumn, Herceptin) 2 orders (2-200 ng on-column, deglycosylated T-DM1) 33
34 Summary 6545XT AdvanceBio QTOF coupled to 1290 infinity II UHPLC provides you: Significant improvement on spectral quality Best sensitivity for intact mab quantitation achieved using standard flow Best linear range Excellent reproducibility on both mass accuracy and response at different levels including the LLOQ level Accurate measurement of glyco-form relative abundances across the linear range including the LLOQ level 34
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