Dyes for analysis of protein aggregation

Abstract

Provided are dyes and compositions which are useful in a number of applications, such as the detection and monitoring protein aggregation, kinetic studies of protein aggregation, neurofibrillary plaques analysis, evaluation of protein formulation stability, and analysis of molecular chaperone activity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This is a continuation-in-part application of U.S. application Ser. No. 12 / 592,639, filed Nov. 30, 2009.BACKGROUND OF THE INVENTION[0002](1) Field of the Invention[0003]The present application generally relates to dyes and compositions comprising dyes. More particularly, provided are dyes and compositions for identifying and quantifying protein aggregation.[0004](2) Description of the Related Art[0005]The deposition of insoluble protein aggregates, known as amyloid fibrils, in various tissues and organs is associated with a number of neurodegenerative diseases, including Alzheimer's, Huntington's and Parkinson's diseases, senile systemic amyloidosis and spongiform encephalopathies (Volkova et al., 2007; Stefani & Dobson, 2003). Fibrillar deposits with characteristics of amyloid are also formed by several other proteins unrelated to disease, including the whey protein beta-lactoglobulin (BLG). All amyloid fibers, independent of the protein...

AI Technical Summary

Benefits of technology

These dyes provide a robust, high-throughput method for detecting protein aggregates, enhancing the accuracy and sensitivity of protein stability analysis, and enabling the monitoring of aggregation kinetics, thereby improving the formulation and storage stability of therapeutic proteins.

Problems solved by technology

Structurally altered proteins have an especially strong tendency to aggregate, often leading to their eventual precipitation.

Irreversible aggregation is a major problem for the long-term storage and stability of therapeutic proteins and for their shipment and handling.

In the bioprocessing arena, the mechanisms of protein aggregation are still not fully understood, despite the fact that aggregation is a major problem in therapeutic protein development (Arakawa et al., 2006).

If the contaminant is the damaged protein itself, then its aggregation may lead to soluble oligomers, which become larger aggregates, visible particulates, or insoluble precipitates.

Damaged forms of a protein product can also arise from chemical modification (such as oxidation or deamidation) and from conformationally damaged forms arising from thermal stress, shear, or surface-induced denaturation.

Further, reversible self-association of proteins can significantly alter overall pharmaceutical properties of product solutions, such as solution viscosity.

Detection of reversible aggregates can be an especially challenging task.

One consequence of the complexities of monitoring aggregate formation processes is the difficulty of linking the effect (presence of aggregates) to its underlying cause, particularly because the key damage may occur at a time or place quite separated from the observed consequence.

Since the earliest clinical applications of protein pharmaceuticals in medicine, aggregation problems have been implicated in adverse reactions in humans and other safety issues.

AUC-SV suffers from lower precision than SEC, however.

Despite its advantages, AUC-SV is not yet readily amenable for use as a routine release test in the biotechnology industry because of issues related to low throughput, the need for specialized equipment, performance problems at high protein concentrations, the need for skilled practitioners of the method, and difficulty in validating data analysis software.

This method is highly sensitive to large aggregates because the intensity of scattered light increases proportionally with molecular weight.

Although this method is ideal for detecting very low mass fractions of large aggregates, it cannot resolve species that are similar in size.

DLS is also not amenable to use as a control method because it is semi-quantitative and very sensitive to dust or other extraneous particles.

When compared with SEC, the precision and limit of detection of aFFF is inferior in the high-molecular-weight range, because of increased baseline noise.

Experimental conditions (e.g., cross-flow rate) for reasonable separations in one size range are also not generally applicable to other size ranges, making the technique cumbersome, especially when analyzing a broad range of masses.

Along with other limitations, such as the need for specialized equipment and a skilled operator, and the difficulty in validating the method prevents the use of aFFF in applications for release and stability monitoring.

SEC cannot handle a large range of sizes because the pore size or degree of polymerization of the resin must be adjusted to the size of the protein species.

If a protein sample contains widely different sizes, many techniques are unsuitable for analyzing all sizes simultaneously.

FFF and DLS can cover a very large range of sizes, but in the case of DLS, resolution is generally fairly poor, and FFF entails some trade-off between resolution and dynamic range.

The resolution of SV-AUC is generally not ideal for separating monomer from dimer, compared with the best SEC columns (especially for lower molecular weight proteins).

The quantification of species that elute from SEC or FFF is quite good, but aggregates can easily be lost during the separation process.

Thus, SEC and FFF may provide good precision but poor accuracy.

For SV-AUC, loss of protein aggregates to surfaces is usually not a problem, but accurate quantification of small oligomers (dimer-tetramer) at total levels of ˜2% or less is quite difficult.

DLS is for all practical purposes useless for detecting oligomers smaller than an octamer, because the technique cannot resolve such oligomers from monomeric species, and for those protein aggregate species that are resolved, the accuracy of the weight fractions is quite poor, typically plus or minus factors of two to ten.

Overall, no single analytical technique is ideal for every protein or is optimal for analyzing the wide range of aggregation problems that can arise with protein pharmaceutical formulation.

Amyloid fibril detection assays have suffered from several drawbacks, however, when using thioflavin T, Congo red and their derivatives.

Furthermore, the binding of dyes can influence the stability of amyloid aggregates, and the interplay with other components (for example, during testing of potential amyloid inhibitors) is unpredictable (Murakami et al., 2003).

Despite the widespread use of thioflavin T, its application to amyloid quantification often generates inconsistent and inaccurate results.

Variations in spectral properties caused by buffer conditions and protein-dye ratios result in poor reproducibility, complicating the use of thioflavin T for quantitative assessment of fibril formation.

When the delivery device is worn close to the body or implanted within the body, a patient's own body heat and body motion, plus turbulence generated in the delivery tubing and pump, impart a high level of thermo-mechanical stress to a protein formulation.

In addition, infusion delivery devices expose the protein to hydrophobic interfaces in the delivery syringes and catheters.

These interfacial interactions tend to destabilize the protein formulation by inducing denaturation of the native structure of the protein at these hydrophobic interfaces.

The formulation of protein drugs is a difficult and time-consuming process, mainly due to the structural complexity of proteins and the very specific physical and chemical properties they possess.

The conventional analytical methods usually require a long period of time to perform, typically twenty or more days, as well as manual intervention during this period.

The development of new formulations is costly in terms of time and resources.

Moreover, even for a known protein formulation, batch to batch quality control analysis is often less than optimal using the current state of the art methods.

This type of technology usually requires a high protein concentration, therefore, it is not cost-effective.

In addition, thermal shift technology cannot work effectively on formulations with low protein concentrations or finalize protein formulations which require a very low detection limit (typically ˜1-5% protein aggregates).

One of the major complications encountered in both in vitro and in vivo protein folding is aggregation resulting from the commonly encountered low solubility of the unfolded protein or different folding intermediates.

Upon heating, BLG aggregates, which can be accelerated by subjecting the protein to either an elevated pH or through the additional of DTT. α-crystallin prevents heat-induced BLG aggregation, acting as a chaperone in the absence of DTT; in the presence of DTT, however, this chaperone activity is less efficient due to faster aggregation of heated and reduced beta-lactoglobulin.

Images