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

Screening Proteins for Manufacturability

Technology Permits Characterization and Preformulation Studies

  • Applications

    Click Image To Enlarge +
    Figure 2. Effect of increasing amounts of sorbitol on IgG physical stability: (A) Effect on thermal conformational stability of IgG in pH 4 buffer, note the significant increase in Tm indicating increased thermal stability with increasing sorbitol concentration. (B) Effect on thermally induced aggregation, note the increase in Tagg but little significant effect on rate of aggregation. (C) Effect on acid induced unfolding, note the shift in peak position to shorter wavelength at 25ºC with increasing sorbitol indicating increased population of the native state.

    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. 



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