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Tutorials : Nov 15, 2010 (Vol. 30, No. 20)

Screening Proteins for Manufacturability

Technology Permits Characterization and Preformulation Studies
  • Simon Webster, Ph.D.

The complex 3-D structure of a protein ultimately determines its biological function. As such, maintaining the native conformation is clearly of critical importance if the protein in question is to be used as a therapeutic. Additionally, loss of higher order structure through complete unfolding or generation of partially folded intermediates can lead to rapid, irreversible aggregation, which can lead to problems ranging from loss of material during production to a raised immune response in patients.

The higher order structure of many proteins is susceptible to partial or total unfolding caused by a range of stresses including temperature, pH, or shear forces. The biopharmaceutical developer, therefore, needs to devise strategies to limit or prevent unfolding and aggregation when proteins are inevitably exposed to stresses during manufacture, storage, and use.

Although protein stability and aggregation resistance can be enhanced through the use of a range of additives at the formulation stage, there is a compelling argument to select or design a fundamentally stable and aggregation-resistant protein as early as possible during the development process. Trying to progress a fundamentally unstable protein can be fraught with difficulty, risk, and cost.

Unfortunately, however, it is often the case in early-stage development that there are many candidate molecules with acceptable affinity for their targets but that the candidate protein, time, manpower, and other resources are all in short supply. This can severely restrict the extent of experimental work that can be performed and consequently limit the amount of information upon which to base informed decisions about which candidate to take forward.

Physical-Stability Screening

In order to provide biopharmaceutical developers with more information at an earlier stage low sample volume or scale-down analytical technologies are required. The Optim 1000 developed by Avacta Analytical has been designed to provide information about conformational stability and aggregation propensity with high throughput and using small amounts of protein, with the added benefit of fully automated operation, data analysis, and reporting.

The development of a specialized sample holder or microcuvette array (MCA) allows the system to analyze up to 48 samples in one run using as little as 1 µL of protein solution in each sample. Depending on the sample and application this can mean using concentrations to below 0.1 mg/mL, corresponding to 100 ng of protein per sample.

The MCA permits high-quality fluorescence and light-scattering data to be recorded from these small volumes, and thermoelectric heating and cooling allows the samples to be subjected to a thermal ramp or held at a fixed temperature during analysis.

In the basic instrument the samples are analyzed by two techniques simultaneously: intrinsic protein fluorescence spectroscopy is used to monitor protein tertiary structure and static light scattering is utilized to monitor protein aggregation. In addition, extrinsic fluorescent dyes can be added to the sample to give complementary information about protein conformation and aggregation. These analytical techniques are particularly well suited to rapid, sensitive analysis of small volumes of protein in the widest range of possible buffer or solvent conditions.

In a typical experiment to elucidate stability and aggregation propensity, the protein samples are heated while their intrinsic fluorescence and light-scattering signals are simultaneously monitored. Figure 1 shows typical output of such an experiment.

Changes in the fluorescence emission (Figure 1A) show the loss of higher order structure and that the protein is thermally denatured. By monitoring this signal, Optim can determine a temperature midpoint, Tm, of the unfolding transition (Figure 1B). The scattered light intensity increases strongly when aggregates are formed and this can be characterized using a second parameter, the aggregation onset temperature, Tagg (Figure 1C).

These parameters can be used to screen different candidate proteins or explore the effect of different buffer conditions and additives on protein structure and aggregation. The data generated is comparable to that produced by alternative technologies such as differential scanning calorimetry, circular dichroism, and dynamic light scattering, but Optim is typically much faster and uses orders of magnitude less protein.

Applications

This technology can be applied to the screening of candidate molecules at the preformulation stage of development. The extremely low sample requirements and the rapidity of the measurements means that the conformational stability and aggregation propensity of the candidates can be explored in a much wider range of environments than was previously possible at this early stage.

A typical preformulation screen might investigate 96 different solvent environments that are selected to be representative of those experienced during development, manufacture, storage, and use as a biopharmaceutical.

For example, conditions such as those experienced in chromatography operations and with elution buffers, low pH buffers such those used during a viral-clearance step, reverse-phase chromatography mobile phases, and common final formulation buffers could all be investigated. With Optim such a study takes less than a working day to run all 96 samples, approximately one hour of analyst time, and as little as 10 µg of protein.

Some of the results from a simple study  in which the effect of increasing amounts of sorbitol on an IgG was investigated are shown in Figure 2. The data in Figure 2A shows that sorbitol increases the thermal conformational stability of the protein. This is reflected by the shift in Tm, the thermal mid-point of the unfolding transition, to higher temperature as the sorbitol concentration is increased.

Sorbitol also raises the Tagg of the antibody (Figure 2B), although the rate of aggregation and, qualitatively, the final degree of aggregation does not appear to be modulated by the addition of sorbitol. In this study, the effects of acid unfolding as well as thermal unfolding were investigated, and Figure 2C indicates that sorbitol stabilizes the antibody against acid unfolding.

The multidimensional nature of protein stability and aggregation data generated from extensive matrices of sample conditions requires adept reporting tools. The pH and temperature dependence of protein-conformational stability can be represented in a conformational stability map making a comprehensive characterization dataset quite accessible to the user.

Conclusion

Scale-down analytics are key to providing early-stage characterization of proteins to allow improved, rational decision making and assist in delivering a quality by design approach to biopharmaceutical development.

The Avacta Optim 1000, is capable of rapidly obtaining high-quality information about higher order structure, stability, and aggregation from small sample volumes. It is applicable to a wide range of protein characterization challenges such as those encountered in preformulation, formulation development, and comparability studies.