综述专论

多角度静态光散射技术表征生物大分子影响因素

  • 胡川梅 ,
  • 吕晶 ,
  • 严翠霞 ,
  • 邵泓 ,
  • 郑璐侠
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  • 1.上海市食品药品检验研究院, 上海 201203;
    2.国家药品监督管理局治疗类单抗质量控制重点实验室, 上海 201203
第一作者 Tel:(021)50798176;E-mail:hcmyjs@163.com
*Tel:(021)50798176;E-mail:zhengluxia@smda.gov.cn

收稿日期: 2024-10-08

  网络出版日期: 2025-11-13

Review on the factors influencing the characterization of biological macromolecules by multi-angle static light scattering technology

  • HU Chuan-mei ,
  • LÜ Jing ,
  • YAN Cui-xia ,
  • SHAO Hong ,
  • ZHENG Lu-xia
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  • 1. Shanghai Institute for Food and Drug Control, Shanghai 201203, China;
    2. NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies, Shanghai 201203, China

Received date: 2024-10-08

  Online published: 2025-11-13

摘要

多角度静态光散射技术是生物大分子领域重要的表征手段之一,能够获得生物大分子丰富的结构信息,如分子量、分子量分布、均方根旋转半径、第二维里系数、分子链构象等,但该技术原理较为复杂,表征的准确性受多种因素影响。本文介绍了生物大分子稀溶液中多角度静态光散射的基本原理,针对该技术准确表征的影响因素,从dn/dc、第二维里系数(A2)、延迟体积、外推形式、荧光干扰等方面进行文献综述和经验总结。本文可以帮助分析工作者更好地理解并掌握这一技术,以获得准确的表征数据。

本文引用格式

胡川梅 , 吕晶 , 严翠霞 , 邵泓 , 郑璐侠 . 多角度静态光散射技术表征生物大分子影响因素[J]. 药物分析杂志, 2025 , 45(7) : 1157 -1165 . DOI: 10.16155/j.0254-1793.2024-1182

Abstract

Multi-angle static light scattering (MALS) is a critical characterization technique in the field of biological macromolecules, providing comprehensive structural information such as molecular weight, molecular weight distribution, root mean square radius, second dimensional coefficient, molecular chain conformation, etc. However, the complexity of its underlying principles and multiple influencing factors often challenge the accuracy of its measurements. This review focuses on the basic theory of multi-angle static light scattering in dilute solutions of biological macromolecules and the analysis of its influencing factors in the characterization of dilute solutions of biological macromolecules including dn/dc value, second dimensional coefficient (A2), interdetector delay volume, extrapolation models, and fluorescence interference. By addressing these variables, this work aims to assist analysts in better understanding and mastering multi-angle static light scattering technology to obtain reliable and precise characterization data.

参考文献

[1] LORD RAYLEIGH FRS.On the calculation of the frequency of vibration of a system in its gravest mode,with an example from hydrodynamics[J].Philos Mag Series,1899,47(289):566
[2] BERNE BJ,PECORA R.Dynamic Light Scattering:With Applications to Chemistrys,Biology,and Physics[M].New York:Dover Publications,Inc.,2000:5
[3] 邹剑,杨蕾,林涛,等.SEC-RI-MALLS测定甘露聚糖肽分子量与分子量分布[J].药物分析杂志,2024,44(2):256
ZOU J,YANG L,LIN T,et al.Determination of molecular weight and distribution of mannatide by means of SEC-RI-MALLS[J].Chin J Pharm Anal,2024,44(2):256
[4] DENG JZ,LIN J,CHEN M,et al.Characterization of high molecular weight pneumococcal conjugate by SEC-MALS and AF4-MALS[J].Polymers(Basel),2022,14(18):3769
[5] MCINTOSH NL,BERGUIG GY,KARIM OA,et al.Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering[J].Sci Rep,2021,11(1):3012
[6] GENTILUOMO L,SCHNEIDER V,ROESSNER D,et al.Coupling multi-angle light scattering to reverse-phase ultra-high-pressure chromatography(RP-UPLC-MALS)for the characterization monoclonal antibodies[J].Sci Rep,2019,9(1):14965
[7] AMARTELY H,AVRAHAM O,FRIEDLER A,et al.Coupling multi angle light scattering to ion exchange chromatography(IEX-MALS)for protein characterization[J].Sci Rep,2018,8(1):6907
[8] WYSOR SK,MARCUS RK.In-line coupling of capillary-channeled polymer fiber columns with optical absorbance and multi-angle light scattering detection for the isolation and characterization of exosomes[J].Anal Bioanal Chem,2024,416(14):3325
[9] MARASSI V,CASOLARI S,RODA B,et al.Hollow-fiber flow field-flow fractionation and multi-angle light scattering investigation of the size,shape and metal-release of silver nanoparticles in aqueous medium for nano-risk assessment[J].J Pharm Biomed Anal,2015,106:92
[10] WYATT PJ.Light scattering and the absolute characterization of macromolecules[J].Anal Chim Acta,1993,272(1):1
[11] PODZIMEK S.The use of GPC coupled with a multiangle laser light scattering photometer for the characterization of polymers.On the determination of molecular weight,size and branching[J].J Appl Polymer Sci,1994,54(1):91
[12] RUSSO PS,STRELETZKY KA,HUBERTY W,et al.Characterization of polymers by static light scattering[M]//Molecular Characterization of Polymers.Amsterdam:Elsevier,2021:499
[13] WU H.Correlations between the Rayleigh ratio and the wavelength for toluene and benzene[J].Chem Phys,2010,367(1):44
[14] Wyatt Technology Corporation.ASTRA for Windows User’s Guide[Z].Version 4.90.2002
[15] LANG P.Scattering Methods:Basic Principles and Application to Polymer and Colloidal Solutions.Summer Term Notes.Part I.[EB/OL].[2023-03-28].https://www.yumpu.com/en/document/read/33367130/basic-principles-and-application-to-polymer-and-colloidalsolutions
[16] GRIGORAS AG.Investigation of cellulose-based materials applied in life sciences using laser light scattering methods[J].Polymers,2024,16(8):1170
[17] PODZIMEK S.Truths and myths about the determination of molar mass distribution of synthetic and natural polymers by size exclusion chromatography[J].J Appl Polymer Sci,2014,131(7):40111
[18] BRANDRUP J,IMMERGUT EH,GRULKE EA,et al.Polymer Handbook.Vol.89[M].New York:Wiley,1999:87
[19] BALL V,RAMSDEN JJ.Buffer dependence of refractive index increments of protein solutions[J].Biopolymers,1998,46(7):489
[20] HELD D.Tips & Tricks GPC/SEC:How to Determine dn/dc Values[EB/OL].(2015-10-04)[2015-10-04].https://www.chromatographyonline.com/view/tips-tricks-gpcsec-how-determine-dndc-values.
[21] MATET B,CRÉPET A,FAYE C,et al.Molar-mass analysis of dendrigraft poly(l-lysine) (DGL)polyelectrolytes by SEC-MALLS:the “Cornerstone” refractive index increment[J].Macromol Chem Phys,2015,216(1):95
[22] MHATRE R,KRULL IS.Determination of on-line differential refractive index and molecular weight via gradient HPLC interfaced with low-angle laser light scattering,ultraviolet,and refractive index detection[J].Anal Chem,1993,65(3):283
[23] COTO B,ESCOLA JM,SUÁREZ I,et al.Determination of dn/dc values for ethylene-propylene copolymers[J].Polym Test,2007,26(5):568
[24] BEAN SR,LOOKHART GL.Factors influencing the characterization of gluten proteins by size-exclusion chromatography and multiangle laser light scattering(SEC-MALLS)[J].Cereal Chem,2001,78(5):608
[25] WANG WJ,KHARCHENKO S,MIGLER K,et al.Triple-detector GPC characterization and processing behavior of long-chain-branched polyethylene prepared by solution polymerization with constrained geometry catalyst[J].Polymer,2004,45(19):6495
[26] ITAKURA M,SATO K,LUSENKOVA MA,et al.Molecular weight dependency of refractive index increment of polystyrene determined by uniform oligomers[J].J Appl Polym Sci,2004,94(3):1101
[27] HAN Y,LI D,LI D,et al.Impact of refractive index increment on the determination of molecular weight of hyaluronic acid by muti-angle laser light-scattering technique[J].Sci Rep,2020,10(1):1858
[28] ZHAO H,BROWN PH,SSHUCK P.On the distribution of protein refractive index increments[J].Biophys J,2011,100(9):2309
[29] LAVRIC S,PREIS J,ROSENAUER C,et al.Determination of molar masses of macromolecules by size exclusion chromatography-light scattering not requiring knowledge of refractive index increments[J].
J Chromatogr A,2017,1520:103
[30] STRIEGEL AM.Absolute molar mass determination in mixed solvents.
2.SEC/MALS/DRI in a mix of two “nearly-isovirial” solvents[J].Anal Chim Acta,2022,1231:340369
[31] THITIRATSAKUL R,BALKE ST,MOUREY TH.Combining detectors in size exclusion chromatography:Ⅰ.Interdetector volume[J].Int J Polym Anal Charact,1996,2(4):345
[32] NETOPILÍK M,PODZIMEK Š,KRACHVÍI P.Determination of the interdetector volume by s-detection in size-exclusion chromatography of polymers with on-line multiangle light-scattering detection[J].J Chromatogr A,2004,1045(1-2):37
[33] NETOPILÍK M.Analysis of the influence of interdetector volume on local calibration in size exclusion chromatography with dual multiangle-light-scattering/concentration detection[J].J Chromatogr A,2006,1113(1-2):162
[34] MATSUMOTO Y,KIKUCHI M,UEDA K,et al.Highly reliable determination of the interdetector delay volume in SEC-MALS for precise characterization of macromolecules having narrow and broad molar mass distributions[J].Polym J,2023,55(3):239
[35] PODZIMEK S.Light Scattering,Size Exclusion Chromatography and Asymmetric Flow Field Flow Fractionation:Powerful Tools for the Characterization of Polymers,Proteins and Nanoparticles[M].New York:Wiley,2012:1857
[36] WITTGREN B,WAHLUND KG.Size characterization of modified celluloses in various solvents using flow FFF-MALS and MB-MALS[J].Carbohydr Polym,2000,43(1):63
[37] KLEIN M,MENTA M,DACOBA TG,et al.Advanced nanomedicine characterization by DLS and AF4-UV-MALS:application to a HIV nanovaccine[J].J Pharm Biomed Anal,2020,179:113017
[38] JUNA S,WILLIAMS PA,DAVIES S.Determination of molecular mass distribution of amylopectin using asymmetrical flow field-flow fractionation[J].Carbohydr Polym,2011,83(3):1384
[39] ANDERSSON M,WITTGREN B,WAHLUND KG.Accuracy in multiangle light scattering measurements for molar mass and radius estimations.Model calculations and experiments[J].Anal Chem,2003,75(16):4279
[40] SSTRIEGEL AM,TRAINOFF SP.Entropic-based separation of diastereomers:size-exclusion chromatography with online viscometry and refractometry detection for analysis of blends of mannose and galactose methyl-α-pyranosides at “Ideal” size-exclusion conditions[J].Chromatographia,2021,84(1):37
[41] VELOURS C,ZHOU J,ZECCHIN P,et al.Determination of the absolute molar mass of[Fe-S]-containing proteins using size exclusion chromatography-multi-angle light scattering(SEC-MALS)[J].Biomolecules,2022,12(2):270
[42] ZINOVYEV G,SULAEVA I,PODZIMEK S,et al.Getting closer to absolute molar masses of technical lignins[J].ChemSusChem,2018,11(18):3259
[43] CLOBEES ML,KOZLIAK EI,KUBÁTOVÁ A.Advancing molecular weight determination of lignin by multi-angle light scattering[J].Polymers(Basel),2024,16(4):477
[44] Technical Note TN3000,Wyatt Technology,Calibration and Normalization of MALS Detectors[EB/OL].[2022-02-02].
https://chem.uiowa.edu/sites/chem.uiowa.edu/files/groups/TN3000%20-%20Calibration%20and%20Normalization%20of%20MALS%20Detectors%20Rev%20A.pdf
[45] WANG Y,SONG YJ,LI ZH,et al.Measurement of solution properties and molecular weight of hydroxyethyl starches using multi-angle laser light scattering:an interlaboratory comparison[J].J Pharm Biomed Anal,2022,219:114905
[46] HADAR J,GARNER J,SKIDMORE S,et al.Correlation analysis of refractive index(dn/dc)for PLGAs with different ratios of lactide to glycolide[C]//2018 Controlled Release Society(CRS)Annual Meeting.New York,2018:95
[47] EP 8.0[S].2013:857
[48] REN Y,LIU L,LIU Y,et al.USP’s characterization of commercial poly(lactic-co-glycolic acid)utilizing SEC-Multi-Angle light scattering and refractive index techniques via absolute method approach[J].J Pharm Biomed Anal Open,2024,4:100031
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