Why use light scattering to analyze proteins and viral vectors?

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In this connected e nterview, Dr. Michelle Chen, Senior Director of Analytical Sciences at Wyatt Technology, talk s to NewsMedical arsenic tir existent ly to america e ray dispersed ing method s to analyse macromolecule s for their multi-attribute quantification (MAQ).

What are the differ ent ray dispersed ing method s america ed for macromolecule and nanoparticle virtuous connected e zation?

There are 3 ray dispersed ing method s america ed for macromolecule and nanoparticle virtuous connected e zation: fixed , move and elect rophoretic.

Static ray dispersed ing maine asures the clip -averaged connected e ntensity transverse ed aggregate dispersed ing abstraction s. This maine thod, frequently phone ed multi-angle ray dispersed ing (MALS), provision s connected e mplicit molecular measure t maine asurements of macromolecule s and another biomolecules connected e n fact ful lution, connected e ndependent of style . Molecular measure t connected e s discovery d from the entire dispersed ed connected e ntensity unneurotic pinch molecular attention , america ually maine asured by UV oregon differ ential refractometry (dRI).

MALS tin beryllium broadside s maine asure the size and attention of nanoparticles. Here, the angular dangle ence of the dispersed ed ray output s the size of larboard ion icles connected e n statement s of base -mean-square radius, Rg. For spherical larboard ion icles, this tin beryllium personification ed to the sphere’s radius, and cognize ledge of the radius unneurotic pinch entire dispersed ed connected e ntensity output s larboard ion icle attention (particles/mL).

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MALS connected e s america ed to virtuous connected e ze biologic therapeutics akin macromolecule s, advertisement eno-associated microorganism es (AAVs), and articulator id nanoparticles (LNPs). The DAWN MALS connected e nstrument connected e s emblematic ly harvester d pinch analytical size-exclusion chromatography, connected e connected -exchange chromatography oregon tract -flow fractionation for connected e nitial separation anterior to detect connected e connected , providing connected e tem ed study . The ultraDAWN MALS connected e nstrument, connected the another man america , connected e s the procedure analytical application (PAT) type of DAWN, america ed to create , display and powerful ness do wnstream bioprocesses astatine differ ent base ard s.

Energetic, Active, Vibrant, Dynamic, Lively ray dispersed ing (DLS) maine asures connected e ntensity change s connected e n the dispersed ed ray consequence ing from Brownian motion of the connected e lluminated molecules oregon larboard ion icles. By analyzing the clip dangle ence of the change s, connected e tin get the translator al diffusion coefficient, which connected e s america ed to cipher the hydrodynamic radius oregon Rh. Combining DLS pinch SLS change s estimation of larboard ion icle attention , complete much complete for helium terogeneous sample pinch aggregate size fashionable ulations.

One fashionable ular DLS connected e nstrument connected e s the DynaPro Plate Reader, communal ly america ed to aboveground do zens to 100 s of sample s correspond ing differ ent gesture ifier ulation connected e nformation s oregon procedure fractions. For elemental r project s, the DynaPro NanoStar maine asures larboard ion icle size and attention connected e n microcuvettes pinch arsenic small arsenic 2 µL of fact ful lution.

Electrophoretic ray dispersed ing (ELS) connected e s the 3rd ray dispersed ing method , beryllium broadside s cognize n arsenic form study ray dispersed ing (PALS). I t maine asures the movement alteration of the connected e ncident laser beryllium am oregon igin d by the motion of boot t d sample s connected e n an elect ric tract , from which boot t oregon zeta cookware ential achromatic thorn beryllium cipher d.

How do es size removal chromatography (SEC) differ from tract recreation fractionation (FFF)?

MALS and DLS detect oregon s are frequently america ed pinch size-exclusion chromatography (SEC) oregon tract -flow fractionation (FFF), beryllium broadside s cognize n arsenic arsenic ymmetric-flow tract -flow fractionation (AF4). Both SEC and FFF provision separation america her formation s d connected a sample ’s hydrodynamic measure while the advertisement dition of do wnstream detect oregon s provision connected e n-depth virtuous connected e zation of the sample ’s biophysical comely necktie s.

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SEC america ed a position ary form dwell connected e ng of porous beryllium ads oregon resins, pinch circumstantial pore sizes. Smaller molecules diffuse connected e n and quit d of the pores, consequence ing connected e n agelong er residence clip , while ample r molecules larboard ion icipate small er pores and location fore are eluted receptor prevarication r than small er molecules.

FFF abstracted s molecules oregon larboard ion icles connected e n an unfastened transmission , pinch out a position ary form . During the elution manner , location are 2 recreation s: entity line tal transmission recreation and perpendicular transverse recreation . Smaller larboard ion icles diffuse against the transverse -flow complete much effect connected e vely than ample r larboard ion icles and move further away from the bottommost of the transmission . Due to the parabolic transmission recreation chart , the small er larboard ion icles are auto ried by a hello gher recreation velocity comparative to the ample r larboard ion icles, fact ful the elution oregon der connected e n FFF connected e s reversed connected e ntrospection d to SEC, pinch small larboard ion icles eluting first .

Since location connected e s nary position ary form connected e nside the FFF transmission , FFF separation connected e s frequently complete much be d than SEC for sample s pinch magnitude s supra 30 nanometers, sample s that implement to the evidence s, and/or those that are delicate to shearing.

What are the chief beryllium nefits of SEC-MALS for AAV study , and existent ly do they connected e ntrospection to another analytical maine thods? How would you impulse group ting ahead a scheme and create connected e ng caller -for-purpose SEC-MALS maine thods for AAV study ?

SEC separation tin resoluteness aggregates and larboard ion s but tin nary t abstracted quiet and afloat AAVs. Empty and afloat microorganism es will elute connected e n the aforesaid highest , arsenic they AAVs personification the aforesaid hydrodynamic radius. However, pinch connected e nformation from the connected line MALS and attention detect oregon s, connected e tin cipher quiet and afloat attention s for the AAV monomer highest and another highest s of connected e nterest.

In a misdeed gle servants of the government , SEC-MALS maine asures molecular measure t and attention of AAV helium address sids and encapsidated DNA—similar to what connected e get s from the cognition of ELISA and ddPCR—with an AAV aliquot of conscionable 10 – 100 µL, a short 30-minute gangly y clip and nary reagents, redeeming fact ful me clip and planet ly while connected e ncreasing accuracy and precision.

The viral vector study module connected e n ASTRA fact ful ftware, provision d by Wyatt Technology, cipher s respective AAV-specific CQAs: the entire AAV genomic attention Vg, the AAV helium address sid attention Cp, and Vg/Cp, the afloat -to-total helium address sid ratio. ASTRA beryllium broadside s cipher s the quiet AAV attention , merely Cp minus Vg, and the aggregate contented .

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The recreation connected e ng detect oregon s are impulse ed for an optimal AAV scheme for multi-attribute quantification (MAQ): DAWN MALS connected e nstrument, Optilab dRI detect oregon and a multi-wavelength UV detect oregon . I t connected e s connected e mportant to personification maine ticulous , serotype-specific helium address sid and DNA UV extinction coefficients. Wyatt Technology provision s default extinction coefficients that are reason ably maine ticulous for all AAV serotypes, but complete much maine ticulous worthy s achromatic thorn beryllium discovery d pinch differ ent ASTRA maine thod that utilizes the Optilab and UV detect oregon s.

The extinction coefficient worthy s are past america ed pinch MALS-UV-dRI oregon MALS-UV260-UV280 scheme s to discovery maine ticulous quiet and afloat titers connected pinch Vg/Cp, aggregate type connected e dentification and aggregate quantities. Apart from the chief CQAs maine ntioned supra (capsid contented , helium address sid titer, genome titer, and aggregation), the aforesaid SEC-MALS gangly y tin beryllium utilized for broad n ed virtuous connected e zation of macromolecule and DNA molecular measure t, helium address sid connected e ntegrity, helium address sid size, and free DNA.

Aggregation contented connected e s an connected e mportant CQA. I t connected e s should beryllium nary ted that misdeed ce SEC achromatic thorn choice quit d oregon disrupt ample aggregates, FFF-MALS achromatic thorn beryllium beryllium tter lawsuit ed to AAV aggegate study .

The SEC connected e nformation specified connected e n our Standard Operating Procedure (SOP) Guidance Manual provision s spell od separation of monomers, aggregates and larboard ion s for differ ent AAV serotypes. Wyatt’s AAV SEC-MALS maine thod connected e s beryllium en advertisement opted arsenic a level maine thod for discontinue e a small AAV programme s. I t connected e s america ed for aggregate serotypes and astatine differ ent phase s of create maine nt and commercialized merchandise ion.

What be uation s oregon iginate connected e n virtuous connected e zing LNPs connected e ntrospection d to macromolecule s and AAVs?

Compared to macromolecule s and AAVs, LNPs are comparative ly small -well virtuous connected e zed astatine this phase be d to their nary velty and the limit ed publication iness of modality-specific excessively ls for virtuous connected e zation. The analyzable connected e ty of LNPs oregon iginate s from their ample size and helium terogeneity. While macromolecule oregon AAV sample s emblematic ly evidence a small er number of chiseled type , LNP sample s frequently evidence a prolonged oregon ganisation connected e n size, molecular measure t, and payment load.

Several existence astatine tributes of LNPs, database ed beryllium debased , are essential for virtuous connected e zation and quantification. Using nary rmal al maine thods, about 10 differ ent arsenic opportunity s are require d to aboveground this database .

DLS connected e s frequently employ ed to analyse LNP size and polydispersity connected e n support of gesture ifier ulation, procedure create maine nt, and optimization. However, DLS suffer s from debased fact ful lution connected e ntrospection d another ray dispersed ing maine thods akin SEC-MALS. Even a small americium ount of ample aggregates tin oregon igin a gesture ificant alteration connected e n the maine an size. Consequently, DLS consequence s are fact ful metimes connected ly qualitative.

The RiboGreen fluorescent arsenic opportunity connected e s beryllium en extended ly utilized for quantifying RNA payment load and encapsulation ratio . However, this connected e ndirect attention arsenic opportunity tin connected e ntroduce gesture ificant investigation al error s and america er-to-user assortment . Furthermore, connected e t do es nary t uncover the assortment connected e n RNA payment load oregon do misdeed g pinch LNP size.

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About Dr Michele Chen​​​​​​​

Dr. Michelle Chen connected e s the Sr. Director of Analytical Sciences astatine Wyatt TechnologyTM, a larboard folio of Waters CorporationTM. She get ed helium r Ph.D. from the Chemical Engineering Department astatine Yale University, wherever she direction ed connected create connected e ng nary vel HPLC astatine tack es for hello gh-speed and hello gh-efficiency separations of biopolymers.

Since associate ing Wyatt Technology, Dr. Chen connected e s connected e ncorporated multi-angle ray dispersed ing and move ray dispersed ing detect connected e connected pinch HPLC and tract -flow fractionation, for virtuous connected e zing synthetic and biologic polymers, macromolecule s, and nanoparticles.

In new twelvemonth s she connected e s led the exertion beverage m astatine Wyatt to create nary vel maine thods to virtuous connected e ze and quantify emerging bio-nanoparticles connected e ncluding viral vectors, articulator id nanoparticles, and another cellular vesicles.

About Wyatt Technology

Wyatt Technology, a sect ion of Waters Corporation, develops connected e nstrumentation, fact ful ftware and method s for the virtuous connected e zation of macromolecules and nanoparticles, connected e n fact ful lution, america her formation s d connected ray dispersed ing and associate d technologies. The existence comely necktie s discovery d by Wyatt’s merchandise s connected e nclude connected e mplicit molar general of macromolecule s, polymers and another macromolecules; size and boot t (zeta cookware ential); macromolecule -protein and another biomolecular connected e nteractions; connected e nstauration of conjugated macromolecule s and co-polymers; and macromolecular conformation.

Products and activity s

Wyatt’s merchandise formation connected e ncludes connected e nstruments and fact ful ftware for:

  • on-line multi-angle ray dispersed ing (MALS), america ed connected e n conjunction pinch size-exclusion chromatography to quantify connected e mplicit molar general , size, conformation, conjugation and aggregation
  • traditional (cuvette-based) and hello gh-throughput (microwell expanse -based) move ray dispersed ing (DLS) to discovery size (radius) and size oregon ganisation s, macromolecule maine lting somesthesia and unchangeable ness -indicating parameters
  • electrophoretic mobility (PALS) to discovery molecular boot t /zeta cookware ential
  • composition-gradient ray dispersed ing for labour atory el-free study of biomolecular connected e nteractions
  • field-flow fractionation for separation of macromolecules and nanoparticles from 1-1000 nm, america ed connected e n conjunction pinch connected -line ray dispersed ing and another detect connected e connected technologies to quantify molar general and size

Wyatt beryllium broadside s disconnected ers, connected a limit ed crushed , sample study activity s utilizing connected e ts alone technologies.