Protein Quality Control in SPR and BLI High-Throughput Screening Studies
Daniel Some, PhD
Principle Scientist
Wyatt Technology Corp

This article outlines the benefits of the use of high throughput dynamic light scattering for screening and discovery of candidate biotherapeutics .

Surface Plasmon Resonance (SPR) and Bio Layer Interferometry(BLI) are powerful techniques, widely used in the discovery of candidate biotherapeutics. High - throughput screening by SPR and BLI is utilized to rank candidates for advantageous properties such as high affinity for the therapeutic target and rapid binding kinetics. Important considerations in this process, often overlooked, are the purity and solution properties of analytes which can adversely affect the data quality and reliability. 'Dirty' samples can even impact the microfluidic integrity of the instrumentation. Sample quality is therefore critical to optimal candidate selection. High-throughput dynamic light scattering (HT-DLS) effectively evaluates the quality of protein samples prior to screening without compromising the analytes.

SPR and BLI High Throughput Screening

In high-throughput SPR, active compound molecules are immobilized on the surface of a chip and potential binding partner solutions are added at various concentrations to determine affinity and kinetics of interaction between the two substances. In BLI the target molecules are attached to fiber-optic probes which are then immersed into protein samples contained within a microwell plate.

Typical SPR and BLI processes often require testing dozens to hundreds of candidates. The short amount of time involved in these rapid screens is critical in the race to discover the next biotherapeutic. SPR commonly draws samples from microwell plates and injects into microfluidic channels. The analytes flow over the immobilized target molecule; good candidates will be strongly attracted to the target on the surface of the chip as detected by the optical system. A similar process occurs in BLI but no microfluidics are involved. In both cases an optical probe provides a signal proportional to the increase in surface-bound mass. Analysis of these signals over multiple analyte concentrations yields affinity and kinetics.

Importance of Analyte Quality

Impurities of a low molecular weight such as extractables and leachables rarely affect SPR or BLI measurements. However, larger particulates such as aggregates and foreign particles can have an adverse effect on the results of both techniques. These unwanted particulates can give rise to inaccurate experimental assumptions in addition to generating noisy and spurious signals in the recorded data. It is therefore beneficial for researchers to be able to identify poorquality protein solutions before proceeding with screening. Such a quality assessment is absolutely necessary to achieve reliable results and, ultimately, passing down the pipeline the best therapeutic candidates for clinical development.

Evanescent optical fields extend a few hundred nanometers into the solution. It is sufficient for any nanoparticle or aggregate passing within that distance from the surface of the chip or fiber probe to cause a signal spike approximately proportional to its mass. Consider a 100 x 100 μm² surface immobilized with bound ligand and illuminated by the SPR beam. Exposing this surface to a concentration of analyte >> Kd results in full coverage and a maximum binding signal. Now, consider a single large contaminating nanoparticle of ~5 μm diameter in that analyte solution. Since the contaminant contains the same volume as the bound analyte, the contaminant particle can generate a noise spike in the binding signal equivalent to the entire bound analyte, if passing close enough to the surface of the chip. Smaller nanoparticles and aggregates do not generate such noticeable interference in the results, however their presence is known to produce a steady stream of small signal fluctuations leading to a degraded optical response. Both effects are represented in the simulated sensorgrams shown in Figure 1.


Figure 1: Sensorgram obtained in the presence of aggregated analyte (simulated).

The presence of active analyte aggregates in sufficient quantities will generate a larger signal than would be expected of the monomeric-target molecule interaction. This leads to higher affinity readings and on rates which in turn distorts the binding response. Aggregates presenting multiple binding sites may exhibit 'avidity' effects, interacting simultaneously with multiple immobilized molecules or exhibiting a reduced dissociation rate by hop-scotching along the surface of the chip. This too leads to affinity being considerably overestimated. In the case of inactive analyte aggregates , effective concentrations are lower than the measured total concentration, causing an apparent decrease in affinity. As demonstrated in Figure 2, the presence of aggregates, active or inactive, leads to the incorrect quantification of candidate binding properties.


Figure 2: The possible interactions between aggregates and target molecules in SPR and BLI screening.

Due to the complications that arise from the presence of aggregates, standard SPR and BLI analyses require analyte solutions to be monomeric at the concentrations employed in the experiment. Poorly formulated or otherwise 'sticky' analytes may result in self-association and the creation of oligomers, such as dimers or tetramers, and the actual concentration of monomer varies with protein concentration.

Much as aggregated analytes can cause experimental errors, so too can aggregated immobilized proteins. In particular, the presence of protein aggregates on the chip or fiber probe surface can result in a reduction in active material or in the average number of exposed epitopes per immobilized mass. Consequently, aggregated substrate proteins also lead to a significant decrease in the apparent affinity.

Large aggregates and foreign particulates are especially undesirable in multichannel SPR as the long and narrow fluidic channels are prone to plugging by agglomerated proteins or other 'nanocrud'1. The occurrence of clogging during a screen of dozens or hundreds of candidates can result in the invalidation of entire studies involving exhaustive protein expression, purification and preparation. In addition to the loss of work, the recovery of plugged microfluidics can be extremely costly in both lost time and money repairing damaged equipment.

Ensuring Protein Quality with Dynamic Light Scattering

Dynamic Light Scattering (DLS) is a non-invasive, non-perturbative optical technique that provides measurements of the size distribution of nanoparticles in solution/suspension and offers a number of unique benefits for the pre-screening of protein solutions. Relying on the principles of Brownian motion to determine diffusion rates of particles in solution, DLS can resolve size distributions over sizes ranging from less than 1 nm up to several micrometers. Once the data has been transformed by software such as DYNAMICS (Wyatt Technology) into a particle size distribution, this can be evaluated to determine whether the solution may safely be injected into SPR microfluidics and whether the SPR or BLI measurements will produce reliable results.

DYNAMICS provides automated analysis and visualization of DLS results as a heat map indicating good, intermediate, and poor protein quality. The entire process may be completed rapidly prior to loading onto the interaction apparatus simply by transferring the microwell plate into the DynaPro HT-DLS system (Wyatt Technology), running the sample screen, and then--when not contra-indicated--loading the same microwell plate onto the SPR or BLI instrument. Microwells that show low-quality material can then be deselected in the interaction screening protocol, helping to ensure the quality of proteins being screened.

Fast, Easy and Effective Resolution of Protein Quality with HT-DLS

Traditional DLS is conducted manually in a microcuvette, one sample at a time. Though this is a valuable process for certain quality assays, it is not feasible to test the hundreds of candidates that are screened using SPR and BLI processes to identify the most promising candidate. In order to establish a viable pre-screening analysis it is necessary to consider accurate, high-throughput processes that save time and laboratory resources. Highthroughput systems such as the DynaPro Plate Reader II (Wyatt Technology ), allow for the analysis process to be completed accurately in situ and in rapid succession, typically requiring only around 10-30 seconds per well including transition time between wells. In addition, with no fluidics, the DynaPro presents no concern for potential carryover of samples between the wells, and the entire screen is set up to proceed unattended.

Another advantageous feature of the DynaPro is the compatibility of the industry-standard microwell plates used for DLS analysis with SPR and BLI apparatus. Once the initial analysis has been completed, the plate can simply be transferred onto the SPR or BLI instrument, and the samples that do not meet quality standards (as determined by DLS) deselected in the interaction screening protocol. An added benefit of HT-DLS is the determination of the analyte's diffusion coefficient, an important property in certain SPR experiments for identifying mass transfer limitations.

In HTS-DLS applications, DYNAMICS is usually configured to bin the data as a heat map based on poor-, intermediate- and high-quality size distributions according to bin definitions specified by the user. For example, a sample showing a single, narrow peak at a size corresponding to that of the monomeric analyte may be classified as high quality and allowed to proceed to the binding assay with high confidence (Figure 3, red wells). An adjoining sample that shows a broadened monomeric peak indicative of some oligomers, and perhaps low levels of additional particulates tens of nanometers in size, may be classified as intermediate quality and allowed to proceed with a warning flag regarding confidence in the results (Figure 3, blue wells). A sample exhibiting significant particulate content in the micron-size range can be assumed to be either contaminated or highly prone to aggregation and prevented from continuing on to the binding assay (Figure 3, black wells).


Figure 3. Visualization of protein quality as a heat map. Total data acquisition time for 96 wells was < 45 minutes. Data courtesy of Sabin Vaccine Institute and Texas Children's Hospital Center at Baylor College of Medicine.

Conclusion

As with most experimental techniques, the quality of the sample ultimately affects the quality of the results. Selection of candidate molecules with the highest potential for optimal therapeutic effect and patient benefit depends on a reliable target binding screen, as performed with SPR or BLI. In order to ensure accurate results from these processes it is necessary to evaluate the quality of sample solutions prior to testing. Those which do not meet standards must then be removed from the screening process to eliminate questionable data and wasted time, as well as prevent potential clogging in microfluidic channels.

HT-DLS can be seamlessly integrated into existing screening workflows to provide classification on the quality of solutions. The addition of a HT-DLS pre-screen process can prevent much of the uncertainty and productivity loss associated with variable ligand quality, leading to more-reliable binding data and confidence for the final candidate selection.

References:

1) http:/www.chi-peptalk.com/biologicsformulation/
2) Karlsson, R . etal., Methods: a companion to Methods in Enzymology, 6, 99 -110, (1994)