Methods for modeling protein stability

The present invention relates to a method for determining the highest temperature that is suitable for performing accelerated protein stability studies, in particular to a method for modeling real-time protein stability from accelerated stability data generated at said temperature, as well as to methods for determining the shelf-life of Factor VIII and ATIII formulations.

Stability studies are indispensable during the development of protein formulations. They are conducted inter alia to define the optimal storage conditions and expiration date of the final product.

To accelerate stability determinations, protein stability studies are often conducted at elevated temperatures. The key issue in interpreting the results of such accelerated protein stability studies is whether the data from accelerated studies can be extrapolated to those under real-time conditions.

Very often accelerated protein stability studies are performed at 40°C. Extrapolation of protein stability at temperatures different from that is based on an Arrhenius plot, wherein linearity of the Arrhenius plot is assumed. However, since protein stability Arrhenius plots in reality are often not linear at higher temperatures, this method can lead to wrong results.

In this context, Ertel and Carstensen describes the use of a modified Arrhenius relationship for the treatment of simulated accelerated stability data. Further, Hei and Clark describes the estimation of melting curves from enzymatic activity-temperature profiles. Furthermore, Nikolova et al. describes the thermostability and activity loss of the exoglucanase/xylanase Cex from Cellulomonas fimi. Furthermore, Nelson describes the use of the Arrhenius model for stability predictions. Moreover, Waterman and Adami describes the prediction of the chemical stability of pharmaceuticals by accelerated aging. Finally, Garrett describes the prediction of the stability of drugs and pharmaceutical preparations.

Therefore, a need exists in the field to provide a method for determining the highest temperature that is suitable for performing accelerated protein stability studies. Further, a need exists in the field to improve current methods for modeling real-time protein stability from accelerated stability data, in order to achieve more accurate and reliable results.

It is an object of the present invention to provide a method for determining the highest temperature that is suitable for performing accelerated protein stability studies. It is also an object of the present invention to provide an improved method for modeling real-time protein stability from accelerated stability data generated at said temperature. In particular, the methods of the present invention can advantageously identify the linear part of a protein stability Arrhenius plot, and thus the highest temperature that should be appropriate for performing accelerated protein stability studies, by determining the short term stability of a specific protein in temperature stress tests.

Arrhenius plots and methods to establish these are known to a person skilled in the art. Briefly, the reciprocal temperature is plotted against the natural logarithm of the rate constant (ln k) of protein stability loss at that temperature. Arrhenius plots for protein stability often exhibit a linear part and a non-linear part, the latter particularly at higher temperatures. Determining the linear part of an Arrhenius plot can be done visually in a straightforward manner. From the lowest 1/T value of the linear part of the Arrhenius plot, the corresponding highest temperature T can easily be calculated.

Methods for performing accelerated protein stability studies and determining real-time protein stability based on accelerated stability data are well known in the art.

The term "protein" as used herein relates to any peptide, oligopeptide, polypeptide, monomeric protein, multimeric protein or multisubunit protein complex. For the purposes of the present invention, a protein may be isolated from a natural source or recombinantly produced. For example, in certain embodiments, the protein formulation comprises a plasma derived blood protein, such as an immunoglobulin, blood coagulation factor, or other protein found in primate plasma. Non-limiting examples of coagulation proteins, which may be purified from a natural source or expressed recombinantly, include, Factor II (prothrombin), Factor III (platelet tissue factor), Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, von Willebrand Factor (vWF), Antithrombin III (AT III), Furin, and ADAMTS proteins. Non-limiting examples of other proteins found in the plasma of primates, include complement factors , Alpha-1 antitrypsin (A1A), Albumin, and an Inter-alpha-trypsin Inhibitor (IαI). Other protein formulations of interest include antibodies and functional fragments thereof, including without limitation, antibodies or immunoglobulins purified from a natural source, recombinant antibodies, chimeric antibodies, huminized antibodies, and fragments thereof. In a preferred embodiment, the protein is a therapeutic protein.

As used herein, an "antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

Non-limiting examples of antibody formulations include, plasma-derived immunoglobulin preparations, recombinant polyclonal or monoclonal preparations, minibodies, diabodies, triabodies, antibody fragments such as Fv, Fab and F(ab)2 or fragmented antibodies such as monovalent or multivalent single chain Fvs in which the variable regions of an antibody are joined together via a linker such as a peptide linker, and the like. Recombinant antibodies include murine antibodies, rodent antibodies, human antibodies, chimeric human antibodies , humanized antibodies, and the like. In preferred embodiments, the recombinant antibody is a human, chimeric human, or humanized antibody suitable for administration to a human.