Essential Biophysical Characterization: the Light Scattering Toolbox from Wyatt Technology

Biotherapeutics Applications

Essential Biophysical Characterization: The Light Scattering Toolbox from Wyatt Technology

Molar Mass Analysis by SEC-MALS Two light scattering flavors for characterization of Traditional SEC relies on biomacromolecules and the assumptions that both bionanoparticles samples and references Multi-Angle static Light Scattering are identical in terms of: (MALS) measures absolute molar mass and size, in solution, via the scattered n Conformation intensity and its angular dependence. n Density A first-principles relationship links these n Hydrophobicity experimental quantities to the product of the molar mass and concentration. These assumptions are By measuring the concentration and often invalid and lead the intensity of scattered light vs. angle, it is to erroneous molar possible to determine mass values, exemplified by tetrameric hemoglobin. The late retention time molar mass and size, would indicate a much lower molar mass for Hb than BSA, even though these independently of elution time or molecular refer- two proteins in fact have very similar molecular weights. MALS provides ence standards. accurate molar masses (dots in graph). MALS is most often utilized in conjunction with a separation technique such as MALS determines molar mass entirely independently of elution time, size-exclusion chromatography for accu- resulting in the correct analysis of Hb. The decreasing molar mass rate characterization of biomacromole- values at late elution times identified by MALS are due to hemoglobin’s cules and bionanoparticles, fragments, oligomers, aggregates and conjugates. well-known tetramer-dimer dissociation (see Hb’s asymmetric peak). scattering volume laser

polarization θ

SEC-MALS Instrumentation for Molar Mass & Size detector

Wyatt’s DAWN® HELEOS® or miniDAWN™ TREOS® MALS detectors interface Dynamic Light Scattering (DLS) with any HPLC-SEC/GPC to create a robust SEC-MALS system for measuring measures the rate of fluctuations of the scattered intensity in order to determine molar mass from 200 Da - 1 GDa, and radii from 10-1000 nm. An optional diffusion coefficients. These are convert- integrated DLS module (WyattQELS™) offers simultaneous measurement of ed to a measure of size hydrodynamic radii from 1-300 nm. known as the hydrodynamic radius. By making The system is rounded out with assumptions about the molecular conformation ® ™ an Optilab T-rEX refractive it is possible to estimate index (RI) detector, which serves molar masses.

7 8 9 4 5 6 DAWN 1 2 3 HELEOS-II DLS offers certain advantages with as a sensitive, highly linear, 0 . TAB ESC DEL universal concentration detector ENTER respect to MALS: it provides coarse size distributions even without separation, and WyattQELS that does not depend on is amenable to high-throughput screening in microtiter plates. Therefore it is useful chromophores or fluorophores. 7 8 9 4 5 6 Optilab 1 2 3 T-rEX 0 . for quick assessments of size, aggrega- TAB ESC DEL

ENTER tion and quality. DLS excels at screening changes in size and aggregation state due to formulation buffer, excipients, concentration and temperature. The Light Scattering Toolbox Enhanced separation and SEC-MALS APPLICATIONS analysis of macromolecules and nanoparticles: FFF-MALS Field-Flow Aggregate Detection by SEC-MALS Fractionation (FFF) is a very useful separation In addition to determining accurately the molar mass and size of aggregates technique for biopoly- mers and nanoparticles. separated by SEC, MALS is exceptionally sensitive to small quantities of large Wyatt’s Eclipse™ FFF aggregates. This exquisite sensitivity derives from the relationship between system offers many scattered intensity and the product of concentration and molar mass: as the benefits: n molecular weight of an aggregate increases, proportionally less and less Large dynamic range: 1-1000 nm n aggregate is needed for detection. Macromolecules: Separation as good as, or better than, SEC n Nanoparticles: Ultra-high resolution 1.0 IgG separations 0.025 dimer n IgG Versatility: Separation can be 0.020 307 266 monomer optimized for different size ranges 0.015 234 188 kDa simply by adjusting flow parameters 0.010 0.5 and/or a spacer 0.005 n Relative Scale Solvent flexibility: Works with most 0 IgG fragment aqueous solvents 1.6 1.8 2.0 2.2 2.4 n Volume (mL) Low shear: FFF utilizes an open 0.0 channel with no stationary phase

1.0 1.5 2.0 2.5 3.0 n Volume (mL) Ideal separation: Adverse surface interactions are greatly reduced The nested plots show the ability to detect and quantify quite small n Productivity: Wyatt’s selection of amounts of aggregates in the IgG UHPLC-SEC chromatogram. The FFF channels separate nanograms to milligrams of sample molar masses of the peaks correspond to oligomers of IgG monomer and fragments. An FFF-MALS system combining a DAWN HELEOS II MALS detector with an Eclipse constitutes a powerful tool for characterizing complex fluids comprising sub-micron Conjugate Analysis by SEC-MALS particles and macromolecules. FFF is recognized by the FDA as an orthog- Proteins conjugated to polysaccharides, glycans or surfactant micelles are onal technique to SEC for aggregates. impossible to characterize correctly by standard SEC, since there are no Asymmetric-flow FFF representative reference standards. A combined SEC-UV-MALS-RI system separation principle: serves to determine the molar masses of the protein and modifier compo- Cross flow gently pushes nents individually, as well as Insect & Mammalian particles the combined molar mass. against the Conjugate analysis makes it semipermeable bottom mem- possible to establish the oligo- 100 brane, while meric nature of a conjugated Protein Mw Brownian motion causes smaller particles Carbohydrate to diffuse back towards the center of the protein or a membrane protein Mw 10 Carbohydrate Mw channel. solubilized in detergent. Even Molar Mass (kDa) Mammalian Channel flow sweeps the particles in the drug-antibody ratio (DAR) Cell Insect Cell parallel to the membrane. The parabolic of antibody-drug conjugates 1 velocity profile of the channel flow may be measured. 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 increases the flow rate of smaller Time (min) particles relative to larger particles: small particles elute first. SEC-MALS indicates that the proteinaceous component of this complex is identical whether expressed in insect or mammalian cells. However, Cross Flow the degree of glycosylation differs greatly. SEC-MALS conjugate analysis using a Wyatt MALS and RI detector with ASTRA® software provides Diffusion the degree of glycosylation and heterogeneity across each eluting peak. Parabolic Frit Channel Flow Membrane The Light Scattering Toolbox Biomolecular interactions: CG-MALS for label-free, FFF-MALS APPLICATIONS immobilization-free analysis

Remove SEC or Whole Serum Analysis Virus-Like Particles GPC fractionation, and MALS measures Not only are the numbers of LDL and The development of VLP-based bio- weight-average molar mass Mw. This proper- HDL particles important indicators of therapeutics depends on FFF-MALS ty is utilized in Wyatt’s coronary risks, but also the size of LDL to assess complete assembly, hetero- Calypso® composi- particles. LDL particles smaller than geneity and even payload. FFF may tion-gradient multi-angle light scattering (CG-MALS) system to characterize the Kd 25.8 nm cause arterial blockage. FFF- be tuned for excellent resolution, as of dynamic equilibria. Because CG-MALS MALS identifies the molecular weight in this example of VLP separation measures molar masses directly, it excels and size and of each serum component. showing a bimodal population. at establishing the molecular stoichiometry of association, i.e. the degree of oligo- mer that forms, or whether a complex is 1:1, 2:1 or 2:2. Few biophysical interaction techniques offer this extended capability. Self-association: At solution concentrations well below Kd, a self-associating protein will be essentially monomeric, while at concentrations well above Kd, the protein will be mostly oligomerized. A CG-MALS method automatically prepares and measures a dilution series to track Fragile entities such as liposomes are not disrupted by FFF. Non- changes in Mw. The dependence of Mw encapsulated peptides or oligonucleotides are readily separated on concentration is analyzed to determine from liposomes and quantitated. both affinity and stoichiometry.

trimer, Kd = 10 µm dimer, Kd = 1 µm dimer, Kd = 10 µm The Light Scattering Toolbox dimer, Kd = 100 µm CG-MALS APPLICATIONS

Solution Molar Mass No interaction

Protein Conc. (log scale) Insulin Self-Association Antibody-Antigen Binding Hetero-association: Hetero-interactions The oligomeric state of biotherapeutic CG-MALS can assess self-association are analyzed across a series of mixed compositions. The light scattering vs. insulin is an essential component of its of each protein as well as the binding composition curve is analyzed to stability and efficacy. CG-MALS shows affinity and absolute stoichiometry of determine binding affinity and molecular that Zn-free insulin undergoes indefinite hetero-association. No self-association stoichiometry, even for multi-valent complexes or simultaneous self- and self-association with K = 52 µM. The was detected in streptavidin-IgG binding, d hetero-association. data are not fit by a monomer-hexamer though hetero-association is evident. Concentration of Species B association model. Kd = 1 nM Antibody Hetero-association Thrombin Kd = 10 nM Gradient Gradient Gradient K = 100 nM 45 4 d 40 Measured Light Scattering ) 35 Best fit isodesmic model 3 No interaction

(kD a 30 Best-fit monomer-hexamer

Mw 25 2 t Light Scattering Signal Peak height gives interaction strength 20 e n r Concentration of Species A 15 1

App a 10 Concentration of Species B 5 0 3:1 0 20 40 60 80 100 120 140 Peak position gives

0 Light Scattering Signal (kDa*g/L) 0.01 0.1 1 10 Time (min) stoichiometry 2:1 Insulin Concentration (mg/mL) Actual LS Signal LS Signal If No Interaction 1:1 While CG-MALS is most Solution binding kinetics appear in No interactio often used to measure n the data, though are not used for Kd. Light Scattering Signal equilibrium binding, it also Concentration of Species A provides rates of aggrega- Analysis in the CALYPSO software For more information, read: Some, D. Light tion, self-assembly and finds Kd = 9 nM with an absolute Scattering Based Analysis of Biomolecular dissociation. molecular stoichiometry of 2:1. Interactions, Biophys. Rev. 5(2) pp. 147-158 The Light Scattering Toolbox Bibliography of Light DLS APPLICATIONS Scattering Publications To search our free, extensive bibliography of over 12,000 peer- DLS serves as an effective and practical tool for formulation screening: it reviewed publications citing Wyatt’s determines aggregation state and propensity for aggregation via conforma- light scattering instruments, visit www.wyatt.com/Bibliography. tional and colloidal stability indicators simultaneously while requiring very little sample per condition. On-Demand Webinars Aggregation as a consequence of applied stress (here freeze-thaw) indicates and Application Notes Would you like to learn more about the the effect of different 20 excipients on stability. many uses of MALS and DLS for the MAB1 PBS pre-lyophilized biophysical characterization of proteins, Thermal stability can be 15 MAB1 PBS lyophilized peptides, oligonucleotides, complexes, MAB2 Sucrose 2% pre-lyophilized vesicles and other biomacromolecules? quantified by ramping y MAB2 Sucrose 2% lyophilized Wyatt offers multiple educational channels temperature to identify 10 including training courses and a freely the onset of aggregation,

% Intensit available library of on-demand webinars or holding at fixed temp- 5 and application notes. erature to measure the Visit www.wyatt.com/Webinars to view 0 these and other on-demand webinars: rate of aggregation. 1.0 10 100 1000 n Radius (nm) VLP Characterization: the Light Scattering Biophysical Toolbox

n The DynaPro® Plate Reader II utilizes industry- To SEC-MALS & Beyond - The Road to standard microtiter plates with 96, 384 or 1536 wells Biotherapeutic Development Illuminated by Light Scattering to measure DLS over 4-85ºC, with read times as n short as 5 sec/well. Screening & Characterizing Biomolecular Interactions via Light Scattering: Part 1 – High Throughput Screening by DLS and Part 2 – CG-MALS n Automated Zeta Potential to Characterize Linker-Induced Instability in ADCs the Charge of Proteins and Particles Conjugating the same monoclonal antibody and drug via different linkers can Visit www.wyatt.com/AppNotes to read recent application notes such as: have significant impact on stability, as shown in this example. ADC2 exhibits two n pH Effects on Stability and Homogeneity thermal transitions, one at 60ºC, similar to ADC1, while the other is near 50ºC. of a Protein Complex DLS highlights the impact of thermally-induced aggregation, negligible in ADC1 n Screening High-Concentration Protein- yet rapid and extensive in ADC2. Protein Interactions n Identification of Insulin Oligomeric States ADC ADC 7.0 1 600 2 n Membrane Protein Quaternary Structure Analysis and Detergent Selection 400 n Au Nanoparticles in a Complex Biological T = 59.8ºC T = 63.9ºC onset,2 Matrix 6.0 onset n Radius (nm) Radius (nm) 200 Multi-Domain Interactions in the Bacterial Tonset,1 = 48.6ºC Flagellar Motor Complex

n 0 Competitive Binding Between Thrombin, 20.0 40.0 60.0 20.0 40.0 60.0 Antithrombin III, and a Monoclonal Temp (ºC) Temp (ºC) Anti-Thrombin Antibody n Charge and Interaction Analysis for Dynamic light scattering is an essential biophysical characterization tool Predicting Antibody Formulation Stability for assessing protein quality, conformation, oligomeric state and stability, with as little as 1-2 µL of sample. Learn the Theory Visit www.wyatt.com/Theory to learn more about the underlying science of multi-angle and dynamic light scattering. 6330 Hollister Avenue Santa Barbara, CA 93117 Tel: +1 (805) 681-9009 Fax: +1 (805) 681-0123 © Wyatt Technology Corporation Web: www.wyatt.com W1700C