Development of In - Vitro Techniques which can Measure Changes in the Tertiary Structure of Proteins and Correlating these Changes to In- Vivo (Animal - based) Potency

Abstract

Proteins are very large and complex molecules. Its three dimensional structure determines its functional characteristics. Only a small part of the structure participates in bioactivity and in some cases a few amino acids. Proteins also have many post-translational modifications which are quite heterogeneous even when produced by a single expression clone. These differences are even more magnified with minor variation in the process used for making them. In short, process determines the product characteristics, especially in terms of its functions. Therefore, merely showing that the primary structure is the same is not sufficient to prove that the protein is physiologically active and the activity is the same from batch to batch. The best information on the tertiary structure is therefore provided by bioassays that truly reflect their functional properties. For example, human growth hormone (hGH) shows growth promotion in animals (in-vivo) as well as of cultured cells (in-vitro). The in-vitro assays for some proteins are able to pick up variation in the efficacy of the molecule and therefore closely reflect the in-vivo function. However, not always in-vitro assays can replace the in-vivo assays. This often leaves animal-based testing as the best option for looking into potency. However, with better understanding of how the proteins bind to receptors on the target cells and how they transmit the signal, more cell-based assays are beginning to be accepted as study models for potency estimation. Often some proteins exert their in-vivo biological effect through complex interactions with receptors present on different celltypes often involving different epitopes, and therefore a single cell-based in-vitro assay is not enough to capture the overall biological effect of a therapeutic protein. Therefore, it is necessary to have orthogonal in-vitro assays that collectively can be used to evaluate the “in-vivo” potency without actually carrying out in-vivo animal testing. Present invention focuses on a few in-vitro assays that are designed to reflect subtle variation in the three dimensional structure of proteins. These variations are then checked for trends in the biological activity of the molecule. A positive trend that is parallel and in proportion with the biological activity is the objective behind developing the in-vitro assay. xvi It is believe that the carbohydrate component and the degree of sialyation of glycoproteins are responsible for transmitting different physiologic signals to the target cells. The degree of sialylation of a recombinant glycosylated protein is often affected by the conditions used in expressing the recombinant protein, and therefore the heterogeneity of sialic acid content is dictated by culture conditions. Sialylation imparts a negative charge on the glycoprotein molecule giving rise to many different isoforms. All these isoforms have different half-lives and consequently different pharmacodynamic behavior. The heterogeneity in the degree of sialyation is assayed by the in-vivo bioassay in animal models. For example - the Steelman-Pohley bioassay which uses a rat model is applied for Follicle Stimulating Hormone. Similarly a mice assay is used for estimation of potency for Erythropoietin and Darbepoietin. Such in-vivo assays have limited precision, require large numbers of laboratory animals and involve cumbersome procedures for data generation and interpretation thereby lacking in high throughput. On the other hand an in-vitro cell-based assays offer high sample throughput, higher accuracy and precision compared to any animal-based in-vivo assay. As a proof of concept, an in-vitro cell based report gene assay will be developed which will simulate the rat ovarian weight augmentation assay (Steelman-Pohley bioassay). This in-vitro assay requires generation of a stable cell line that will express the receptors for the target protein (FSH) on the surface of cells. These cells will then respond to varying degrees of proliferation when the receptors are engaged by the target protein. The degree of proliferate will determine the degree of binding which in turn will be dependent on the degree of sialyation. The assay is supposed to mimic the in-vivo bioassay. After establishing a correlation between the in- vivo animal based assay and the in-vitro cell-based proliferation assay, the animal assay can be substituted by the in-vitro method. Such assay can be use because of their simplicity, speed, high-throughput and cost, and will reduce the need of animals.