Quick and effective evaluation of biologics for aggregation and stability is needed to proceed with maximum confidence to the next stages of preclinical development and clinical trials.
In this interview, Dan Some, from Wyatt Technology Corporation, leaders in the production of innovative light scattering instruments, talks to AZoNano about the newly launched all-in-one platform for performing high-throughput screening of biologics to determine their key biophysical attributes.
How can light scattering analysis techniques be used throughout the R&D process?
In R&D it is critical to apply the right analytical tools to answer the questions or meet the analytical challenges presented at each stage. Light scattering is one of the primary analytical technologies used and there is a suite of light scattering analytical tools available to meet a range of specific needs.
In the early stages, evaluation and ranking of small quantities of multiple candidates is needed to make sure that the research is on the right track. This involves screening many different clones, different buffer conditions, et cetera, in order to find the most promising.
Light scattering helps to determine essential solution properties, such as molecular weight and size, quality and stability indicators such as propensity for aggregation, colloidal and thermal stability, solubility, and the viscosity of concentrated protein solutions.
Once the most promising candidates or buffers have been identified, these samples are tested under accelerated stresses, such as freeze/thaw or stirring, shaking, elevated temperatures. The few samples passing all these tests go on to be studied for long-term shelf life stability.
Light scattering is useful to verify important properties, such as the integrity of the drug candidate, and identify and quantify break-down products, such as aggregates or fragments. It can also highlight subtle modifications to the molecule that will show up through protein-protein interactions, and/or the overall net charge.
The final stage of the R&D cycle is to assess and understand what made a particular molecule or buffer fail or succeed. In the context of aggregation or viscosity, this usually means analyzing intermolecular interactions, such as excipient protein binding, interactions between co-solutes in a cocktail of antigens or therapeutic monoclonal antibodies, the likelihood of self-association, and the molecular charge or zeta potential that contribute to these interactions. Light scattering provides the means to answer these questions.
How do the various light scattering technologies differ in terms of what they can measure?
There are two central aspects of analytical light scattering.
The first is static light scattering (SLS), which can measure from first principles the molar mass of a molecule in solution. This can help to identify a protein and determine its oligomeric or aggregate state and analyze the extent of glycosylation.
Measuring how molar mass changes with concentration or composition facilitates analysis of interactions, and in particular protein-protein interactions. For example, to determine the equilibrium dissociation constant K
d for specific interactions, or the second virial coefficient A 2 for non-specific interactions.
The second, dynamic light scattering (DLS) takes advantage of the rapid fluctuations in scattered intensity that arise due to diffusion by Brownian motion. Measurement of the rate of fluctuations means that the diffusion coefficient can be determined, which can be converted to the hydrodynamic radius (R
h) that is a measure of size.
Relating the diffusion coefficient to concentration allows another indicator of non-specific protein-protein interactions to be calculated — the diffusion interaction parameter (k
Which analyses are carried out using DLS and which by SLS?
DLS indicates size and size distributions, whereas SLS determines molecular weight.
In a solution containing particles of a single size, DLS can be used to determine the average radius. In a distribution analysis, there would be a single narrow peak. If the solution contains particles with a small size range, for example: monomers, dimers, and trimers, DLS cannot resolve the individual species. There would still be a single peak, but it would be broader. The width of the peak is presented as the polydispersity.
However, if the solution contains several populations of particles and the average radii of each population differs by a factor of three to five, DLS will produce separate peaks. This is known as a multimodal distribution. Each peak, or each mode, will have its own polydispersity representing the range of sizes within that population.
In the context of proteins, this might mean a main peak representing the monomers, dimers and trimers, and a secondary peak representing large aggregates.
SLS will determine molar mass, and provides the weight-average molar mass. This is the average of all the species present in solution and is a first principles measurement.
Both SLS and DLS are really sensitive to aggregates, so they are useful to alert whether there are any aggregates present. Since SLS only provides an average molecular weight, it cannot identify the aggregates present.
Larger aggregates will show up immediately in DLS analysis and smaller aggregates will change the average size although they will not be identified directly by the DLS measurement.
Using a combination of SLS and DLS facilitates differentiation between the unfolding of a protein and aggregation. If there is a change in size (shown by DLS) but not molecular weight (shown by SLS) this indicates unfolding.
On the other hand, if there is an increase in molecular weight, this is a direct indication that aggregation has occurred.
How can light scattering in well plates help in the evaluation of R&D candidates?
There are many, many applications of DLS in well plates for various areas of drug discovery, nanoparticles, and protein R&D.
As we have just seen, light scattering techniques can readily indicate whether aggregation has occurred and this is a key assessment in the selection of promising biologic drugs. A single platform is now available for carrying out all of the screening measurements.
In a 384-well plate, the wells could be populated with five candidates, each at five different pH values, each of those at five different ionic strengths, and each of those across three replicates, and with nine controls or buffers.
These can be scanned in less than one and a half hours to determine polydispersity.
A key benefit of making DLS measurements, or indeed any measurement, in a plate is that there is no additional sample handling beyond pipetting the sample into the plate.
The DynaPro Plate Reader II is well-established in the biopharmaceutical field for screening proteins using dynamic light scattering (DLS). Wyatt Technology has recently launched the latest generation in this product line — the DynaPro Plate Reader III.
Can you tell us more about the new DynaPro Plate Reader III?
DynaPro Plate Reader III is the newest instrument in the DynaPro Plate Reader product line. This exciting product has new capabilities for measuring SLS, as well as DLS.
The addition of SLS enables the DynaPro Plate Reader III to cover a larger set of biophysical attributes related to molar mass. The previous-generation DLS plate reader already determines size, size distributions, colloidal stability (via the diffusion interaction parameter, k
D), and thermal stability (via the size aggregation rate, and the onset temperatures for size increase, T onset, and melting temperature ,T m).
The latest-generation DLS/SLS plate reader can now also determine molar mass, colloidal stability (via second virial coefficient, A
2) and thermal stability (via the onset temperature for aggregation, T agg). Aggregation rates and temperature profiles are quantified simultaneously using measurements of size and molar mass. The DynaPro Plate Reader III thereby provides a good illustration of which aggregates form and their conformation and extent.
As in the previous model, DynaPro Plate Reader III incorporates a built-in camera that photographs the bottom of each well, allowing it to be checked for even bubble formation. Consequently, if the autocorrelation function does not look quite right, it is possible to go back and look for a possible explanation.
The camera images also indicate precipitation, formation of crystals, and turbidity induced by exceeding the solubility limit. Together with the solution properties, the photographs can provide a comprehensive picture of the behavior of samples.
The DynaPro Plate Reader III continues to use standard 96, 384, and 1536 well plates, which means it is possible to test various concentrations and pure buffers, and still include the statistical replicates required to validate the data. Sample volumes can be as low as 4 microliters per sample in a 1536 well plate or up to 150 or 200 microliters in the larger well plates.
It also includes a temperature control feature, facilitating temperature ramps from room temperature up to 85 degrees, and a new evaporation control system to facilitate measurements over a longer term and at elevated temperatures using sealing tape rather than oil capping of wells.
The DynaPro Plate Reader III can be integrated with existing plate-based robotics. Measurements can be made directly in the well plates and the plates then transferred, without perturbation, to another instrument with full automation.
A multi-sample, multi-temperature protocol, can be programmed and completed without intervention.
Importantly, the DynaPro Plate Reader uses a long wavelength laser at 830 nanometers so there is no exposure to ultraviolet light, which carries the risk of causing aggregation in proteins.
How are dynamic light scattering and static light scattering measured in samples in a microplate?
In the DynaPro Plate Reader the plates are illuminated from below with a laser beam. The scattered light coming back out the bottom of the plate then enters an avalanche photodiode.
Most common avalanche photodiodes can make the rapid, high-speed measurements that are needed to make DLS analyses, but they don't have the linearity that's needed to make an effective SLS measurements. In contrast, the DynaPro Plate Reader III includes a new linearized avalanche photodiode, which has an extended range for linearity to facilitate accurate measurement of molecular weights over a good range.
What measurement capabilities does the DynaPro Plate Reader III have?
The new capabilities of the DynaPro Plate Reader III enable measurement of molar mass across an entire plate. Size is measured simultaneously and color coding of the final results can be used to highlight size, molar mass or polydispersity, giving a quick overview of which buffers and which concentrations are most appropriate.
The software includes various types of algorithms allowing programmable filtering based on data quality. For example, if there is some dust in one of the wells, a measurement will not be reported.
The final results for all the measurements, such as molar mass, radius, polydispersity, and the conditions in each well can be exported into spreadsheet format. The data can then easily be combined with information from other analysis techniques to create a comprehensive picture of sample properties and behavior.
Colloidal stability analysis, which is made through the concentration dependence of either the molar mass or the hydrodynamic radius, is perhaps one of the most critical and useful predictors of long-term stability because it indicates directly the protein-protein interactions that lead to aggregation.
The DynaPro Plate Reader III has the ability to measure both A
2 and K d simultaneously with high throughput. There is therefore no need to make do with just measuring K d and calculating a rough approximation of A 2.
The DynaPro Plate Reader III is really an invaluable platform as it rapidly provides a significant section of the biophysical attribute landscape. It gives accurate determinations of size and size distributions, determines the solution molecular weight, including aggregates, the rate of—or propensity for—aggregation, and indications of thermal and colloidal stabilities.
Furthermore, it can analyse dozens of samples, thousands of formulation and varying temperature combinations, and simultaneously provide multiple biophysical attributes, all in familiar, convenient and inexpensive well plates.
Where can our readers go to find out more?
To find out more please visit our site
www.wyatt.com/platereader About Dan Some
Dan Some is responsible for scientific and technical marketing content at Wyatt Technology, ensuring that the scientific community in the field of macromolecules and nanoparticles is aware of the applications and benefits of Wyatt’s advanced instrumentation.
He also studies the current needs of these researchers to help determine future product development at the company.
Previously Dr. Some worked in R&D, developing Wyatt’s Calypso system for characterizing protein-protein interactions.
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