病毒及病毒样颗粒中分子量检测方案(色谱检测器)

LenS3MALS DetectorApplication Note TOSOH BIOSCIENCE LLC ·3604 Horizon Drive， Suite 100， King of Prussia， PA 19406·Tel： 800-366-4875 · email： info.tbl@tosoh.com·www.tosohbioscience.comAN125 0421 Molecular Weight Determination of VLPs Using LenS3Multi-Angle Light Scattering [Detector Viruses and virus like particles (VLPs) are multimeric protein structuresthat mimic native viruses but are non-infectWiaouSs. VLPs are subjects ofinterest， as their potential continues to grow as candidates in newvaccines and gene therapy products. For example， commerciallyavailable VLP-based vaccines are available for Hepatitis B and humanpapillomavirus. Robust analytical techniques are needed to not onlyensure quality of final products but provide data for informeddecision-making during the development process. Size exclusion chromatography (SEC) is an analytical technique thatprovides results on the size and purity of macromolecules. Whencoupled with multi-angle light scattering (MALS)， it offers bothmolecular weight (MW) and radius of gyration (R， or size). Importantly，AU2go detection is only concentration dependent， whereas MALScorresponds to both concentration and molecular weight. Thus， the largemolecular weight characteristic of VLPs inherently provides MALS witha strong scattered light response and enables VLP detection even in adilute solution that is well below AU280 detection limit. The primary challenge in the analysis of very large macromoleculesby SEC is the selection of the appropriate analytical column. Here weexplore TSKgel PWxL series of SEC columns， which include a widerange of different pore sizes on a polymethacrylate stationary phase，for their utility in the analysis of large macromolecules such as VLPs.The protein calibration curves (Figure 1) show the separation range ofTSKgel PWxL columns. The majority of VLPs have a molecular weight of>1 mega Daltons， which make the TSKgel G5000PWxL (100 nm pore size)，TSKgel G6000PWxL (>100 nm pore size) and TSKgel GMPWxL (mixed bed)ideal columns of choice for analysis ofVLPs. Figure 1. Protein calibration curves on TSKgel PWxL columns Columns： TSKgel GMPWxl， 13 pm， 7.8 mm ID×30 cmTSKgel G5000PWxL， 10 pm， 7.8 mm ID×30 cmInstrument： Thermo Scientific UltiMate@3000Mobile phase： 0.145 mol/L NaCl，0.01 mol/L HEPES，0.05% sodium azide， pH 7.4(refractive index， 1.333)Flow rate： 0.3 mL/min or as indicatedDetection： UV： UltiMate 3000 multiple wavelength detectorRl： Shodex RI-504 semi-micro Rl detectorMALS： LenS3 MALS detectorSample： Parvovirus VLP (MVM-MVP) (CygnusTechnologies)， stock 1×102 particles/mL(10-15 pLinjection)， (dn/dc=0.19， dA/dc=N/A)MALS calibrant： BSA， 5 mg/mL(dn/dc =0.185， dA/dc 0.66) In this application， parvovirus VLP was separately analyzed on both aTSKgel GMPWxL and TSKgel G5000PWxL SEC column coupled with theLenS3 MALS detector. Either Rl or UV can function as the concentrationdetector. Rl was used with the right angle light scattering signal (RALS)to measure MW. Extreme low angle (LALS)， right angle， and extremehigh angle (HALS) signals were used to plot angular dissymmetry andto determine R. The MALS detector was calibrated with BSA priorto sample analysis and all data were processed and analyzed usingSECview@ software. Analysis of parvovirus VLP by SEC-MALS using the TSKgel GMPWxLcolumn revealed a MW of ~4 mega Daltons and R， of 12.8 nm (Figure 2).「hese results closely align with reported values for this VLP (Biotech.Prog. 34， 1213-1220，2018). Figure 2. Analysis of parvovirus VLP and BSA on TSKgel GMPWxL mixedbed pore size SEC column As seen in Figure 3， parvovirus VLP was diluted up to 64-fold andinjected at 10 pL onto a TSKgel G5000PWxL column. Approximately3×1010 particles per mL can still be detected using the RALS signal fromthe LenS3 MALS detector， which allows for analysis of materials with lowconcentration or when working with limited sample. Figure 3. Limit of detection by RALS using TSKgel G5000PWxL at 0.5 mL/min Conclusion Mass spectrometry is the most common method previously used forVLP size determination， but this technique is costly and impractical forfrequent analysis. Inclusion of SEC-MALS as an analytical technique todetermine the MW and R， is a preferred alternative and allows for bothroutine analysis and process monitoring. The wide range in pore sizesand separation ranges of TSKgel PWxL SEC columns overcomechallenges in analytical SEC where separations of large macromoleculesrequire a larger pore sized stationary phase. When these SEC columnsare then combined with the greatly enhanced sensitivity of TosohBioscience's LenS3 MALS detector， fast and easy analysis of MWand Rg with an improved level of detection (LOD) is provided. TSKgel and Tosoh Bioscience are registered trademarks of Tosoh Corporation UltiMate is a registered trademark of the Dionex CorporationLenS is a registered trademark of Tosoh Bioscience LLC in the US， India and Japan.SECview is a registered trademark of Tosoh Bioscience LLC in the USA， EU， and India；and of Tosoh Corporation in Japan. 本应用介绍了使用TSKgel PWXL系列尺寸排阻色谱柱并配合Tosoh Bioscience的超高灵敏度光散射检测器LenS3使用，可以快速且简便地对病毒样颗粒的MW和Rg进行高水平分析。