Dynamic Light Scattering

With the brand-new Prometheus Panta
ApplicationsTechnology

The new gold standard for stability and particle characterization of biologics

The power of DLS (Dynamic Light Scattering): Thermal unfolding, isothermal and temperature-dependent particle sizing, solution homogeneity and aggregation analysis on one high-end platform.

All DLS and nanoDSF parameters that can be measured with Prometheus Panta are listed below:

nanoDSF

THERMAL UNFOLDING


Tm (for 330 nm, 350 nm, and ratio)
Melting temperature, or point at which 50% is unfolded


Tonset (for ratio)
Temperature at which unfolding begins


Ea (*parameter derived from data)
Activation energy of unfolding


Reversibility of unfolding
Point at which irreversibility happens when unfolding

Dynamic Light Scattering (DLS)

SIZE ANALYSIS


Tsize (from growth of rH in cumulants fit)
Temperature at which average particle size begins to increase


Tscattering (from scattering intensity)
Fragments, PROTACs, ions, nanoparticles, peptides, and carbohydrates


Average scattering intensity
Identifies if concentration is too high or low for proper size distribution analysis

 

CUMULANT OR REGULARIZATION ANALYSIS


rH
Hydrodynamic radius shows size of particle in solvated state


PDI
Polydispersity index represents distribution of size populations

 

SELF-INTERACTION ANALYSES


kD
Diffusion interaction identifies onset of unfolding and impact on colloidal stability


D0 determined from the kD analysis
Theoretical diffusion constant at concentration = 0

Backreflection

AGGREGATION


Tturbidity
Onset temperature of aggregation

Use DLS across your entire biologics workflow

Benfit from the single-platform Prometheus Panta DLS for your biologics from early development and design over validation to formulation and production. Benefit from 2bind’s vast experience in handling and testing all kinds of biologics with biophysical methods. We don’t only get you data, we provide results.

Antibody engineering

Design antibodies, scFv, Fab fragments and any other antibody variant directly with high thermodynamic stability and be confident on which candidates to more forward to the next development stage.

(Pre-) formulation

Test for the important stability attributes of biologics in early pre-formation of candidate molecules as well as development of final clinical formulation conditions.

Process development

Monitor biologics during purification, scale-up, and production and ensure proper stability parameters.

"
Use DLS to
– Determine aggregation propensity
– Get self and non-specific interactions
 
And in combination with nanoDSF to
– Characterize conformational (thermal) stability
"
Use DLS to
– Determine aggregation propensity
– Perform buffer and excipient screening
– Characterize particle sizing and size distribution (homogeneity)
– Determine particle size distribution
– Do this isothermal and across temperature gradients
 
And in combination with nanoDSF to
– Characterize conformational, thermal stability
"
Use DLS to
– Determine aggregation propensity
– Characterize particle size distribution during scale-up and optimization
 
And in combination with nanoDSF to
– Characterize conformational (thermal) stability

Biologics development

Identify suitable candidate molecules based on stability, size, and solution properties as early on as possible and exclude ones with undesirable physical and chemical properties.

Small molecule QC

Test impurity profiles, batch comparability, long-term storage conditions, environmental conditions for investigational products during IND and NDA stages.

Comparability studies

Comparability is key when a manufacturing process is changed. Ensure that changes don’t impair the quality, safety and efficacy of a drug. Tets stability and solution properties pre- and post-change.

"
Use DLS to
– Determine aggregation propensity
– Get self and non-specific interactions
 
And in combination with nanoDSF to
– Characterize conformational (thermal) stability
"
Use DLS to
– Determine particle sizing on reconstitution, dilution, mixing at initial and final time points
 
And in combination with nanoDSF to
– Get thermal stability and particle sizing from forced degradation and photostability studies
"
Use DLS to
– Determine particle size distribution
 
And in combination with nanoDSF to
– Characterize conformational (thermal) stability
– Including stress conditions across broad concentration ranges

Prometheus Panta DLS Advantages

Time-efficient

– 48 samples in parallel
– high-throughput analysis
– up to 1000’s of samples

High versatility

– biologics
– artificial particles
– 1 nm to 2 µm particles

In-depth analysis

– test sample quality
– analyze ligand binding
– identify conformation changes

P

DLS + nanoDSF

– combine DLS with nanoDSF
– particle sizing/aggregation
– plus particle unfolding

Label-free

– Only 10 µl sample
– no labeling
– no immobilization

1

Wide size range

– nm to µm scale
– 1 kDa to 1 MDa
– biologic and artificial particles

DLS Technology

Overview

DLS stands for dynamic light scattering and is the method of choice for particle sizing and optimization of colloidal sample stability. Dynamic light scattering utilizes the elastic scattering of light (also known as Raleigh scattering) to determine the diffusion coefficients and hence the hydrodynamic radii of macromolecules. This enables the rapid measurement of particles of different types and chemistry in solution and without expensive sample pre-treatment: Proteins, peptides, nucleic acids, viruses, micelles, lipids, liposomes, aggregations, emulsions can be measured.

Consequently, DLS is a great tool for buffer and formulation screening as well as screening of small molecule compound libraries for influence on colloidal protein stability and aggregation tendency.

With the new Prometheus Panta device it is possible for the first time to measure colloidal (DLS) and conformational (nanoDSF/DLS) stability in one experiment with minimal sample volume (10 µl) and time (1.5 hours for 48 capillaries with nanoDSF and DLS data). Thus a broad image of comprehensive protein stability pathways can be obtained from a single experiment.

Technology

When light hits particles that smaller than the wavelength of the light, it is scattered in all directions without loss of energy. This effect is known as elastic light scattering. Due to the diffusion of particles in solution (Brownian motion), the scattered light waves interfere with each other in a constructive and destructive manner resulting in intensity fluctuation over time.

Small particles diffuse faster than large ones and the scattered light intensities therefore change faster in a solution with smaller particles than in a solution with larger ones. A digital autocorrelator function now correlates the intensity fluctuation over time, from which diffusion coefficients can be derived. From the diffusion coefficient, the hydrodynamic radius of the sample particles can be determined using the Stokes-Einstein equation.

Typical applications

Due to the robust detection method and quick analysis pace, Prometheus Panta is very versatile and can be used in a number of contexts:

  • Antibody engineering (determine aggregation propensity, get self- and non-specific interactions)
  • Pre-formulation and formulation development (determine aggregation propensity, determine thermal stability, perform buffer and excipient screening, characterize particle sizing and solution homogeneity)
  • Buffer screening
  • Excipient screening
  • Process development (determine and compare protein aggregation, characterize particle sizing and size distribution during process scale-up and optimization)
  • Biologics development (characterize protein unfolding and aggregation, get self- and non-specific interactions, analyze protein size and homogeneity)
  • Small molecule quality control (determine particle sizes after reconstituion, dilution, mixing, isothermally and across temperature gradients)
  • Comparability studies (determine particle size distribution across different batches, lots, processes, perform stress testing of biologics)

 

For more information on these applications or the DLS analysis services by 2bind, take a look at our Application Database or contact our DLS specialists.

Advantages

DLS in general and with the Prometheus Panta in particular offers a great number of benefits:

  • Short measurement time: 48 samples require only 20 minutes for isothermal DLS measurement or just one hour for thermal ramp measurement. This also enables high-throughput analysis of 100s to 1000s of conditions for (pre-)formulation development.
  • Great sample type versatility: All kinds of macromolecular and polymeric sample types can be measured. Sizes down to 1 nm and up to 2 µm can be reliably detected: Peptides, proteins, nucleic acids, lipids and liposomes, artifical polymers, detergents and detergent micelles, viruses and virus-like particles, cell organelles.
  • Deep information content: Get insights into sample quality, abundnat species distribution, effects of ligand binding and conformational changes in one experiment.
  • Combined analysis of colloidal and conformational stability: Combine the forces of classical DLS and nanoDSF protein unfolding analysis to shed light on the most important pathways of protein dysfunctionalization.
  • Label free detection: Only 10 µl sample is required, no sample labeling or immobilization neccesary.
  • Wide molecular weight range: Particle sizes from 1 kDa to 1 MDa.

Frequently asked questions

FAQ – General

What are typical applications for the DLS Technology? Is screening possible?

DLS is a very versatile technique since it can be applied to all kinds of biological particles. Proteins are usually the main target for DLS analysis. Another major class of DLS experiments is development of solution characteristics, for example for biologics formulation development or cosmetics.

Due to the fast analysis pace and high throughput, DLS is well suited for screening of buffers, buffer additives or small molecule influence on protein stability and protein unfolding.

How many samples can be measured in parallel?

With our DLS setup, it is possible to measure 48 samples in parallel.

Is it also possible to analyze the stability and unfolding of protein with DLS?

Normaly not. But our Prometheus Panta instrument combines the ultra-high resolution of DLS with state-of-the art nanoDSF (nano-scale differential scanning fluorimetry) for simultaneous measurement of DLS and protein unfolding.

It even allows for detection of multiple domain unfolding and aggregation events. Even changes in the DLS properties of individual protein domains during unfolding can be monitored, using the DLS regularization fit during thermal melting of a protein.

Is it possible to observe ligand-induced changes of the hydrodynamic radius?

Yes, it is possible to observe the effect of ligand binding on the hydrodynamic radius by monitoring the cumulant radius of the target in the presence of increasing concentrations of the ligand.

Can I characterize protein-protein interactions with DLS?

Yes, it is possible to measure protein-protein interactions with DLS. For this, a continuous variation experiment can be performed, in which the concentrations of the two interaction partners are simultaneously and continuously varied and the mean particle size is determined. Upon binding, the largest particle size will be observed. Note that for this experiment to work, the differences in hydrodynamic radius of the single proteins must not exceed two-fold (or the molecular weight difference must not exceed 5-fold).

FAQ – Samples

How much sample is required?

Prometheus Panta works with ultra-thin glass capillaries. Thus, only 10 µl of sample volume is required per experiment. Even less sample is required, if only isothermal DLS is conducted, since only the sample in the focus area will be measured.

What concentration range of my samples can be measured?

In general, it is possible to analyze protein solutions from 0.1 mg/ml up to more than 20 mg/ml.

What particle size range can be measured?

Particles between 0.5 nm and 2 µm can be measured.

FAQ – Assay Conditions

What is the temperature range for the measurement?

DLS can be measured isothermally at any temperature between 15°C and 95°C. In addition, thermal ramp measurements of DLS and nanoDSF can be done with heating from 15°C to 95°C, with heating rates of 0.1 to 5°C/minute. 

Are there any limitations in buffers or additives?

Our sample preparation protocol carefully prevents any dust or particle contamination prior to measurement. In general, DLS is not limited by buffers or additives. However, it must be noted that every particle in the sample will scatter light. Thus, complex solutions might give background scattering signals that prevents successful DLS analysis. 

Contact Specialist