多肽中保留时间和峰面积的优异重复性检测方案(液相色谱仪)

Protein DigestionSMART Digest Kit (P/N 60109-101) Providing the Highest Retention Timeand Peak Area Reproducibility forMaximal Confidence in PeptideMapping Experiments Mauro De Pra， Ken Cook，2 Mike Oliver， and Carsten Paul"Thermo Fisher Scientific， Germering， Germany2Thermo Fisher Scientific， Hemel Hempstead， United KingdomThermo Fisher Scientific， Runcorn， United Kingdom Key Words Acclaim C18 RSLC， Biocompatible UHPLC， Biopharma，Biotherapeutics Characterization， Monoclonal Antibodies，Protein Digest， Vanquish UHPLC System Goal Provide an ultra-high retention time and peak area precisionexample of the separation of a mAb digest. Introduction Peptide mapping of digested proteins are of high impor-tance when characterizing biotherapeutics. Peptide mapsare utilized to confirm the expression of the intendedamino acid sequence， to confirm genetic stability or toidentify post-translational modifications， especially wheninterfaced with mass spectrometry. Reversed phaseseparation in combination with only UV detection is，however， still very common in stability studies， for inprocess measurements and quality assurance. In thesecases peak areas， peak area ratio and retention timesare sufficient to provide the required information. Forhighest confidence in the qualitative and quantitativeresults of such assays， the retention time as well as thepeak area has to be extremely stable. The Thermo Scientific"VanquishUHPLC systemfeatures a binary pump with extremely low pulsationripple due to a brand new pump concept. In addition，the Vanquish UHPLC system pre-compresses the sampleprior to the injection which results in a highly stable flowdelivery. Thanks to these benefits， the Vanquish UHPLCsystem is capable of providing unmatched retention timeprecision. This retention time precision accompanied witha high peak area precision guarantees the analyticalsuccess for even challenging shallow gradient separationsby a reliable peptide identification and quantification. Inthis work， the separation of peptides obtained from atherapeutic protein is provided. The retention time andpeak area precision is evaluated for repeated injections. EquipmentVanquish UHPLC system consisting of： - Binary Pump H (P/NVH-A10-A) - System Base (P/N VH-S01-A) -Mixer Kit， 200 pL， VH-P1 (6268.5120) - Split Sampler HT (P/N VH-A10-A) -Column Compartment H (P/NVH-C10-A) - Active pre-heater (6732.0110) -Post column cooler， 1 pL (6732.0510) -Diode Array Detector HL (P/N VH-D10-A) -LightPipe" flow cell， standard (10 mm；P/N 6083.0100) Thermo ScientificM DionexChromeleonMChromatogaphy Data System (CDS) software， version 7.2 Experimental Sample Preparation 1. Cetuximab monoclonal antibody (5 mg /mL) wasdiluted 1：4 with the SMART Digest buffer to a finalvolume of 100 pL 2. The diluted sample was then added to a SMART Digesttube and left for 60 minutes at 70°C 3. The digested sample was then centrifuged at 10，000gfor 5 minutes and the supernatant was removed forchromatographic analysis Column： Thermo ScientificAcclaimRSLC 120， C18， 2.2 pm Analytical (2.1×250 mm)， P/N 074812 Mobile Phase： A： 0.05% TFA in water， P/N TFA 85183 B： 0.04% TFA in 8/2 acetonitrile/water (v/v)， P/N acetonitrile TS-51101 Gradient： 0-30 min： 4-50% B， 30-31 min： 50-90% B，31-35 min： 90% B， 35-36 min： 90-4% B， 36-45 min： 4% B Flow Rate： 0.4 mL/min Maximal Pressure： 384 bar Temperature： 80C； Forced Air Mode Injection Volume： 5pl Detection： 214 nm Data Collection Rate： 20 Hz Response Time 0.2 sec Flow Cell： 10mm LightPipeTM Results and Discussion The digestion was achieved utilizing the SMART digestkit. Using this approach the sample preparation timecould be reduced significantly and total preparation timeof the monoclonal antibody (mAb) digest was lower than75 minutes. The separation of the resulting peptides was obtainedwith a 30 minutes gradient， and a total analysis time of45 minutes， including column wash with high organiceluent， and re-equilibration at initial conditions. Figure 1shows the overlay of 13 consecutive injections of the samesample of mAb digest. The results show excellent reproducibility across thewhole chromatogram. On average， standard deviation(SD) was of the order of 0.13 seconds (0.00214 minutes).SD for some peaks was as low as 0.065 seconds； and didnot exceed 0.3 seconds for any peptide. The relative standard deviation was consistently extremelylow (Figure 2). Out of 110 peaks automatically integratedby Chromeleon CDS， 34 had RSD smaller than 0.0100%，reaching the minimum value of 0.0060% for the peak atretention time 23.057 minutes. Please note that the earlyeluting peaks naturally have the highest retention timeRSDs because of a mathematical disadvantage in thecalculation. Figure 1. Overlaid chromatogram of 13 repeated injections of the mAb tryptic digest. Figure 2. Retention Time RSD (%) relative standard deviation measured for13 repeated injections of a mAb digest. In addition， Table 1 gives the peak area reducibility asrelative standard deviation. The relative standard deviation of the peak areas wasbelow 1.0% for all peptides. The average reproducibilitywas 0.4% highlighting the highly reliable sample injectionand peak integration at challenging conditions. Conclusion Stability of retention time and peak areas is critical fora confident evaluation of chromatographic results andto avoid any misinterpretation. The Vanquish UHPLCsystem is extremely reproducible in both retention timeand peak area reproducibility. The retention timeprecision provided by the system enables the analystto deduce any change in retention time to an actualchange of the sample structure. As shown， the peak areareproducibility provided by Vanquish will result in amaximal confidence of quantitative result. Consequently，the Vanquish system meets the requirements of demandingpeptide mapping analysis. Table 1. Peak area of six selected peaks eluting over the entire gradient andspanning a wide concentration range. Retention Time (min) Average Area(mAU*min) RSD Area (%) 5.23 1.04 0.10 10.29 0.36 0.22 13.07 0.05 0.94 15.96 0.96 0.49 22.39 5.17 0.14 24.68 0.25 0.61 SCIENTIFIC AThermo Fisher Scientific BrandANEN