Production of a novel heterodimeric two-chain insulin-Fc fusion protein.

Insulin is a peptide hormone produced by the pancreas. The physiological role of insulin is the regulation of glucose metabolism. Under certain pathological conditions the insulin levels can be reduced leading to the metabolic disorder diabetes mellitus (DM). For type 1 DM and, dependent on the disease progression for type 2 DM, insulin substitution becomes indispensable. To relieve insulin substitution therapy for patients, novel insulin analogs with pharmacokinetic and pharmacodynamic profiles aiming for long-lasting or fast-acting insulins have been developed. The next step in the evolution of novel insulins should be insulin analogs with a time action profile beyond 1-2 days, preferable up to 1 week. Nowadays, insulin is produced in a recombinant manner. This approach facilitates the design and production of further insulin-analogs or insulin-fusion proteins. The usage of the Fc-domain from immunoglobulin as a fusion partner for therapeutic proteins and peptides is widely used to extend their plasma half-life. Insulin consists of two chains, the A- and B-chain, which are connected by two disulfide-bridges. To produce a novel kind of Fc-fusion protein we have fused the A-chain as well as the B-chain to Fc-fragments containing either 'knob' or 'hole' mutations. The 'knob-into-hole' technique is frequently used to force heterodimerization of the Fc-domain. Using this approach, we were able to produce different variants of two-chain-insulin-Fc-protein (tcI-Fc-protein) variants. The tcI-Fc-fusion variants retained activity as shown in in vitro assays. Finally, prolonged blood glucose lowering activity was demonstrated in normoglycemic rats. Overall, we describe here the production of novel insulin-Fc-fusion proteins with prolonged times of action.

A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system.

The drug discovery process in the biopharmaceutical industry usually starts with the generation of plasmids coding for certain proteins. Due to advances in cloning techniques the generation of thousands of different plasmids is not a limiting factor anymore. The next step is the expression and evaluation of the proteins. In recent years significant progress has been made in the miniaturization of protein expression and purification. These processes have been adapted to robotic platforms and hundreds of proteins can be expressed and purified in parallel. As a consequence of miniaturization, the protein purification is restricted to a one-step process. In addition the amount of purified protein is usually in the µg-range. This might be suitable if a sensitive initial screening assay is available. However, when larger amounts of proteins are required robotic platforms are no longer appropriate. In addition, a one-step purification procedure is often not sufficient to obtain pure protein preparations. To address this topic we have used the NGC chromatography system for automated purification of up to five samples using a three-step purification procedure. The first chromatographic step is the capture step followed by a desalting step. The final purification was done using size exclusion chromatography. This set-up reduces the overall-time needed for protein production, needs minimal operator invention, is easy to handle and thus increases the throughput.