Development of in vitro techniques which can measure changes in the tertiry structure of proteins and correlating these changes 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 cell-types 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